Lnc-PLCB1 is stabilized by METTL14 induced m6A modification and inhibits Helicobacter pylori mediated gastric cancer by destabilizing DDX21.

Helicobacter pylori (H. pylori) infection is considered to be an important factor in gastric cancer (GC). Long noncoding RNA (lncRNA) and m6A modification are involved in the occurrence and development of GC, but the role of lncRNA m6A modification in the development of GC mediated by H. pylori is still unclear. Here, we found that H. pylori infection downregulated the expression of lnc-PLCB1 through METTL14-mediated m6A modification and IRF2-mediated transcriptional regulation. Overexpression of lnc-PLCB1 inhibited the proliferation and migration of GC cells, while downregulation of lnc-PLCB1 promoted the proliferation and migration ability of GC cells. In addition, clinical analysis showed that lnc-PLCB1 is lower in GC tissues than in normal tissues. Further study found that lnc-PLCB1 reduced the protein stability of its binding protein DEAD-box helicase 21 (DDX21) and then downregulated the expression of CCND1 and Slug, thereby playing tumour suppressing role in the occurrence and development of GC. In conclusion, the METTL14/lnc-PLCB1/DDX21 axis plays an important role in H. pylori-mediated GC, and lnc-PLCB1 can be used as a new target for GC treatment.

Antibiotic-induced ROS-mediated Fur allosterism contributes to Helicobacter pylori resistance by inhibiting arsR activation of mutS and mutY.

The increasing antibiotic resistance of Helicobacter pylori primarily driven by genetic mutations poses a significant clinical challenge. Although previous research has suggested that antibiotics could induce genetic mutations in H. pylori, the molecular mechanisms regulating the antibiotic induction remain unclear. In this study, we applied various techniques (e.g., fluorescence microscopy, flow cytometry, and multifunctional microplate reader) to discover that three different types of antibiotics could induce the intracellular generation of reactive oxygen species (ROS) in H. pylori. It is well known that ROS, a critical factor contributing to bacterial drug resistance, not only induces damage to bacterial genomic DNA but also inhibits the expression of genes associated with DNA damage repair, thereby increasing the mutation rate of bacterial genes and leading to drug resistance. However, further research is needed to explore the molecular mechanisms underlying the ROS inhibition of the expression of DNA damage repair-related genes in H. pylori. In this work, we validated that ROS could trigger an allosteric change in the iron uptake regulatory protein Fur, causing its transition from apo-Fur to holo-Fur, repressing the expression of the regulatory protein ArsR, ultimately causing the down-regulation of key DNA damage repair genes (e.g., mutS and mutY); this cascade increased the genomic DNA mutation rate in H. pylori. This study unveils a novel mechanism of antibiotic-induced resistance in H. pylori, providing crucial insights for the prevention and control of antibiotic resistance in H. pylori.

Variations in the oral microbiome and metabolome of methamphetamine users.

Drug addiction can seriously damage human physical and mental health, while detoxification is a long and difficult process. Although studies have reported changes in the oral microbiome of methamphetamine (METH) users, the role that the microbiome plays in the process of drug addiction is still unknown. This study aims to explore the function of the microbiome based on analysis of the variations in the oral microbiome and metabolome of METH users. We performed the 16S rRNA sequencing analysis based on the oral saliva samples collected from 278 METH users and 105 healthy controls (CTL). In addition, the untargeted metabolomic profiling was conducted based on 220 samples. Compared to the CTL group, alpha diversity was reduced in the group of METH users and the relative abundances of Peptostreptococcus and Gemella were significantly increased, while the relative abundances of Campylobacter and Aggregatibacter were significantly decreased. Variations were also detected in oral metabolic pathways, including enhanced tryptophan metabolism, lysine biosynthesis, purine metabolism, and steroid biosynthesis. Conversely, the metabolic pathways of porphyrin metabolism, glutathione metabolism, and pentose phosphate were significantly reduced. It was speculated that four key microbial taxa, i.e., Peptostreptococcus, Gemella, Campylobacter, and Aggregatibacter, could be involved in the toxicity and addiction mechanisms of METH by affecting the above metabolic pathways. It was found that with the increase of drug use years, the content of tryptamine associated with neuropsychiatric disorders was gradually increased. Our study provides novel insights into exploring the toxic damage and addiction mechanisms underlying the METH addiction.IMPORTANCEIt was found that with the increase of drug use years, the content of tryptamine associated with neuropsychiatric disorders gradually increased. The prediction models based on oral microbiome and metabolome could effectively predict the methamphetamine (METH) smoking. Our study provides novel insights into the exploration of the molecular mechanisms regulating the toxic damage and addiction of METH as well as new ideas for early prevention and treatment strategies of METH addiction.

Effects of alcohol on the symptoms of gouty arthritis and taxonomic structure of gut microbiota in C57BL/6 mice.

Gout is an acute arthritis caused by the elevated levels of serum uric acid (UA), and its prevalence has been rapidly increasing. Alcohol abuse could lead to a series of health problems. Multiple pieces of evidence suggest that alcohol intake affects the development and progression of gout, while the gut microbiota plays an important role in the development of gout and the long-term alcohol consumption could affect the stability of the gut microbiota. This study aimed to explore the effects of alcohol intake at different concentrations on gouty arthritis based on the gut microbiota. We investigated the effects of different concentrations of alcohol on gouty arthritis in mouse models of acute gouty arthritis established by injection of monosodium urate (MSU) crystals into C57BL/6 mice. The results indicated that the high-alcohol consumption not only exacerbated joint swelling and pain, increased the levels of UA, tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6), but also showed dramatic effects on the composition and structure of the gut microbiota in gouty mice. Two key microorganisms, Parasutterella and Alistipes, could aggravate gout symptoms through lipopolysaccharide biosynthesis, riboflavin metabolism, phenylalanine metabolism, and arginine and proline metabolisms. In conclusion, our study suggested that high-concentrations of alcohol altered the gut microbiota structure in gouty mice induced by MSU crystals, which could exacerbate gouty symptoms by enhancing pro-inflammatory pathways.

SpoT-mediated NapA upregulation promotes oxidative stress-induced Helicobacter pylori biofilm formation and confers multidrug resistance.

Recently, there is increased incidence of drug-resistant Helicobacter pylori infection. Biofilm formation confers multidrug resistance to bacteria. Moreover, it has been found that the formation of biofilm on the surface of gastric mucosa is an important reason for the difficulty of eradication of H. pylori The mechanisms underlying H. pylori biofilm formation in vivo have not been elucidated. Reactive oxygen species (ROS) released by the host immune cells in response to H. pylori infection cannot effectively clear the pathogen. Moreover, the extracellular matrix of the biofilm protects the bacteria against ROS-mediated toxicity. This study hypothesized that ROS can promote H. pylori biofilm formation and treatment with low concentrations of hydrogen peroxide (H2O2) promoted this process in vitro The comparative transcriptome analysis of planktonic and biofilm-forming cells revealed that the expression of SpoT, a (p)ppGpp (guanosine 3'-diphosphate 5'-triphosphate and guanosine 3',5'-bispyrophosphate) synthetase/hydrolase, is upregulated in H2O2-induced biofilms and that knockout of spoT inhibited H. pylori biofilm formation. Additionally, this study examined the key target molecules involved in SpoT regulation using weighted gene co-expression network analysis. The analysis revealed that neutrophil-activating protein (NapA; HP0243) promoted H2O2-induced biofilm formation and conferred multidrug resistance. Furthermore, vitamin C exhibited anti-H. pylori biofilm activity and downregulated the expression of napA in vitro These findings provide novel insight into the clearance of H. pylori biofilms.

The Quinone-Derived Small Molecule M5N32 Is an Effective Anti-Helicobacter pylori Agent Both In Vivo and In Vitro.

BACKGROUND: Helicobacter pylori has become increasingly resistant to all commonly used clinical antibiotics. Therefore, new anti-H. pylori drugs need to be identified. Recently, quinones were found to inhibit growth of H. pylori with quinone-derived small-molecule compounds identified as having antitumor effects. METHODS: The minimum inhibitory concentrations of the compounds against H. pylori were measured by agar plate dilution method. The inhibition of biofilm formation by the compounds was assessed by SYTO9-PI double staining. The reactive oxygen species induced by the compounds were detected by DCFH-DA stain. The clearance effects of the compounds for H. pylori in mouse were evaluated by counting colony-forming units and hematoxylin and eosin staining. RESULTS: Our results revealed strong inhibition of M5N32 in vitro against H. pylori in both the planktonic and biofilm-forming states. Resistance to M5N32 was not developed in successive generations of the bacteria. In vivo, the combination of M5N32 and omeprazole showed enhanced effects in comparison to the standard triple therapy. M5N32 was nontoxic to normal tissues. CONCLUSIONS: M5N32 is effective in the treatment of H. pylori infections, providing potential development of anti-H. pylori medicines in the treatment of H. pylori infections.

CircMAN1A2 is upregulated by Helicobacter pylori and promotes development of gastric cancer.

Helicobacter pylori (H. pylori) is one of the main causes of gastric cancer. It has been reported that circRNAs play a vital role in the development of multiple types of cancer. However, the role of H. pylori-induced circRNAs in the development of gastric cancer has not been studied. In this study, we found that H. pylori could induce the upregulation of circMAN1A2 in AGS and BGC823 cells independent of CagA. The downregulation of circMAN1A2 could inhibit the proliferation, migration and invasion of gastric cancer cells, and circMAN1A2 could promote the progression of gastric cancer induced by H. pylori by sponging miR-1236-3p to regulate MTA2 expression. Furthermore, circMAN1A2 knockdown inhibited xenograft tumour growth in vivo, and the overexpression of circMAN1A2 was associated with the progression of gastric cancer. Hence, Helicobacter pylori induced circMAN1A2 expression to promote the carcinogenesis of gastric cancer, and circMAN1A2 might be a new potential diagnostic marker and therapeutic target for gastric cancer.

Effects of ß-Carotin and Green Tea Powder Diets on Alleviating the Symptoms of Gouty Arthritis and Improving Gut Microbiota in C57BL/6 Mice.

As a chronic metabolic disease caused by disorders of purine metabolism, gout has shown increasing incidence rate worldwide. Considering that gout is not easily treated and cured, further studies are explored to prevent gout development through diet modification. Both ß-carotin and green tea powder are rich in dietary fiber, which helps maintain the balance of gut microbiota in humans. The aim of this study was to investigate the effects of ß-carotin and green tea powder diet on the prevention of gouty arthritis in relation to the bacterial structure of gut microbiota in mice. We successfully induced gouty arthritis in C57BL/6 mice by injecting monosodium urate (MSU) crystals and feeding high-fat diet (HFD), and further investigated the effects of additional ß-carotin and green tea powder in the diets of mice on the prevention of gouty arthritis in mice. Our results showed that diet of ß-carotin and green tea powder reduced the joint swelling and pain in mice with gout, reduced the levels of serum uric acid (UA) and three types of pro-inflammatory cytokines, i.e., interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), improved the gut microbiota profile, and reduced the metabolic levels of purines and pyrimidines. In conclusion, our study provided evidence to support the application of ß-carotin and green tea powder diet as a dietary adjustment method to prevent and treat gouty arthritis.

Helicobacter pylori infection is correlated with the incidence of erosive oral lichen planus and the alteration of the oral microbiome composition.

BACKGROUND: Oral lichen planus (OLP), a common clinical oral disease, is associated with an increased risk of malignant transformation. The mechanism underlying the pathogenesis of OLP is unknown. Oral dysbacteriosis is reported to be one of the aetiological factors of OLP. Although Helicobacter pylori infection is associated with various oral diseases, the correlation between H. pylori infection and OLP is unclear. This study aimed to investigate the effect of H. pylori infection on OLP pathogenesis and oral microbiome composition in the Chinese population, which has a high incidence of H. pylori infection. RESULT: In this study, saliva samples of 30 patients with OLP (OLP group) and 21 negative controls (NC group) were collected. H. pylori infection was detected using the carbon-13-labeled urea breath test (UBT). The saliva samples were divided into the following four groups based on the H. pylori status: H. pylori-positive OLP (OLP+), H. pylori-positive NC (NC+), H. pylori-negative OLP (OLP-), and H. pylori-negative NC (NC-). Oral microbiome compositions were significantly different between the OLP and NC groups and between the OLP- and OLP+ groups. Compared with those in the OLP- group, those in the OLP+ group had a higher incidence of erosive OLP and higher levels of salivary cytokines. In contrast, the oral microbiome composition and cytokine levels were not significantly different between the NC- and NC+ groups. CONCLUSIONS: This is the first report to demonstrate that H. pylori infection is significantly correlated with the pathogenesis of erosive OLP.

Transporters HP0939, HP0497, and HP0471 participate in intrinsic multidrug resistance and biofilm formation in Helicobacter pylori by enhancing drug efflux.

BACKGROUND: The multidrug resistance of Helicobacter pylori is becoming an increasingly serious issue. It is therefore necessary to study the mechanism of multidrug resistance of H pylori. We have previously identified that the HP0939, HP0497, and HP0471 transporters affect the efflux of drugs from H pylori. As efflux pumps participate in bacterial multidrug resistance and biofilm formation, we hypothesized that these transporters could be involved in the multidrug resistance and biofilm formation of H pylori. MATERIALS AND METHODS: We therefore constructed three knockout strains, Δhp0939, Δhp0497, and Δhp0471, and three high-expression strains, Hp0939he , Hp0497he , and Hp0471he , using the wild-type (WT) 26 695 strain of H pylori as the template. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of wild strains, knockout strains, and high-expression strains to amoxicillin, metronidazole, and other antibiotics were measured. The efflux capacity of high-expression strains and wild strains was compared by Hoechst 33 342 accumulation assay. RESULTS: Determination of the MIC and MBC of the antibiotics revealed that the knockout strains were more sensitive to antibiotics, while the high-expression strains were less sensitive to antibiotics, compared to the WT. The ability of the high-expression strains to efflux drugs was significantly higher than that of the WT. We also induced H pylori to form biofilms, and observed that the knockout strains could barely form biofilms and were more sensitive to several antibiotics, compared to the WT. The mRNA expression of hp0939, hp0497, and hp0471 in the clinically sensitive and multidrug-resistant strains was determined, and it was found that these genes were highly expressed in the multidrug-resistant strains that were isolated from the clinics. CONCLUSIONS: In this study, we found three transporters involved in intrinsic multidrug resistance of H pylori.

High-Salt Diet-Induced Gastritis in C57BL/6 Mice is Associated with Microbial Dysbiosis and Alleviated by a Buckwheat Diet.

SCOPE: A high-salt diet is a cause of gastritis, but the associated mechanism remains unclear. Recent studies have shown that gastric flora is associated with a variety of stomach diseases, but it is not known whether gastric flora is involved in gastritis induced by a high-salt diet. METHODS AND RESULTS: Gastritis is successfully induced in C57BL/6 mice fed a high-salt diet (salt: 5% NaCl) for four weeks. Through 16S rRNA gene sequencing, the composition of the stomach microbiota of mice fed normal and high-salt diets are compared, the results of which show that the high-salt diet induces significant changes in the gastric flora. Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) is used to predict the function of the microbiota in the stomach of mice, and the results indicate that a high-salt diet leads to a decrease in the ability of the gastric microbiota to metabolize polysaccharides and vitamins. A buckwheat diet is used to treat gastritis. The results show gastritis induced by the high-salt diet is significantly alleviated, and the dysbiosis in the stomach also improved. CONCLUSION: Buckwheat diet may be one of the ways to prevent and treat gastritis caused by a high-salt diet.

Hydrogen sulfide-mediated resistance against water avoidance stress-induced gastritis by maintenance of gastric microbial homeostasis.

Chronic persistent stress is an important cause of gastritis, but the underlying mechanism remains to be further researched, especially the role of the gastric microbiota in this process. Here, we used the water avoidance stress (WAS) test in mouse models for chronic stress-induced gastritis to investigate the underlying mechanisms of this disease. The effect of stress on the gastric microbiota was analyzed based on 16S rRNA sequencing; the changes in hydrogen sulfide (H2 S) and inflammatory cytokine levels in gastric tissues were detected by Western blotting, ELISA, immunofluorescence, and qRT-PCR. Hematoxylin and eosin staining was used as an indicator of the gastritis histological score. This finding is consistent with previous studies showing that gastric H2 S is negatively associated with the inflammatory index and might protect the gastrointestinal tract from inflammation. WAS-induced gastritis was associated with a reduction in H2 S release, which appeared to affect the homeostasis of the gastric microbiota of mice. Inflammation and microbial dysbiosis were partially reversed by sodium hydrosulfide (NaHS) and vitamin B6 (VB6) supplementation, suggesting the therapeutic potential of VB6 supplementation for the treatment of stress-induced gastritis. Gastritis has a serious impact on health and quality of life. An increasing number of people are suffering from chronic gastritis linked to a high-stress lifestyle, and our research provides clues for the prevention and treatment of stress-induced gastritis.

Downregulation of eukaryotic translation initiation factor 3b inhibited proliferation and metastasis of gastric cancer.

Eukaryotic translation initiation factor 3 (eIF3) plays an important role in the regulation of mRNA translation, cell growth and cancer development. eIF3b is the main scaffolding subunit in the eIF3 complex and has been demonstrated to contribute to the development of several cancers. First, our study found that the downregulation of eIF3b could inhibit the proliferation and metastasis of gastric cancer cells by regulating the expression of cancer-related genes. In addition, the expression of eIF3b correlated with the stage and progression of gastric cancer and was shown to be upregulated in human chronic gastritis and in gastric cancer tissues compared with the expression of eIF3b in normal gastric tissues. Moreover, Helicobacter pylori (H. pylori) infection could upregulate the expression of eIF3b in gastric cancer cells, suggesting that eIF3b might be involved in the carcinogenic process of H. pylori. The above findings identified the oncogenic role of eIF3b in gastric cancer development, and this may contribute to the exploration and discovery of novel therapeutic targets for gastric cancer treatment.

Long noncoding RNA THAP9-AS1 is induced by Helicobacter pylori and promotes cell growth and migration of gastric cancer.

BACKGROUND: Long noncoding RNAs (LncRNAs) have been confirmed to play crucial roles in cancer biology. Gastric cancer (GC) is the third leading cause of cancer related death, and Helicobacter pylori (H. pylori) is the major risk factor for GC. In this study, we focused on the roles of H. pylori-related lncRNAs in the progression of GC. METHOD: Differentially expressed lncRNAs were identified through RNA-seq analysis of H. pylori-infected GC cells. RESULTS: We found that the expression of the lncRNA THAP9-AS1 was up-regulated after infection of GC cells with H. pylori and was higher in GC tissues than in gastritis tissues. Colony formation, CCK8 and transwell assays were executed to show that THAP9-AS1 can promote GC cell proliferation and migration in vitro. Our study identified the pro-oncogenic lncRNA THAP9-AS1, which has a higher expression level in GC tissues than in gastritis tissues and which promoted the proliferation and migration of GC cells in vitro. CONCLUSION: These findings may provide a potential therapeutic target for H. pylori-associated GC.

Research on Path Planning Model Based on Short-Term Traffic Flow Prediction in Intelligent Transportation System.

Vehicle driving path planning is an important information service in intelligent transportation systems. As an important basis for path planning optimization, the travel time prediction method has attracted much attention. However, traffic flow has features of high nonlinearity, time-varying, and uncertainty, which makes it hard for prediction method with single feature to meet the accuracy demand of intelligent transportation system in big data environment. In this paper, the historical vehicle Global Positioning System (GPS) information data is used to establish the traffic prediction model. Firstly, the Clustering in QUEst (CLIQUE)-based clustering algorithm V-CLIQUE is proposed to analyze the historical vehicle GPS data. Secondly, an artificial neural network (ANN)-based prediction model is proposed. Finally, the ANN-based weighted shortest path algorithm, A-Dijkstra, is proposed. We used mean absolute percentage error (MAPE) to evaluate the predictive model and compare it with the predicted results of Average and support regression vector (SRV). Experiments show that the improved ANN path planning model we proposed can accurately predict real-time traffic status at the given location. It has less relative error and saves time for users' travel while saving social resources.

Bifunctional Enzyme SpoT Is Involved in Biofilm Formation of Helicobacter pylori with Multidrug Resistance by Upregulating Efflux Pump Hp1174 (gluP).

The drug resistance of Helicobacter pylori is gradually becoming a serious problem. Biofilm formation is an important factor that leads to multidrug resistance (MDR) in bacteria. The ability of H. pylori to form biofilms on the gastric mucosa is known. However, there are few studies on the regulatory mechanisms of H. pylori biofilm formation and multidrug resistance. Guanosine 3'-diphosphate 5'-triphosphate and guanosine 3',5'-bispyrophosphate [(p)ppGpp] are global regulatory factors and are synthesized in H. pylori by the bifunctional enzyme SpoT. It has been reported that (p)ppGpp is involved in the biofilm formation and multidrug resistance of various bacteria. In this study, we found that SpoT also plays an important role in H. pylori biofilm formation and multidrug resistance. Therefore, it was necessary to carry out some further studies regarding its regulatory mechanism. Considering that efflux pumps are of great importance in the biofilm formation and multidrug resistance of bacteria, we tried to determine whether efflux pumps controlled by SpoT participate in these activities. We found that Hp1174 (glucose/galactose transporter [gluP]), an efflux pump of the major facilitator superfamily (MFS), is highly expressed in biofilm-forming and multidrug-resistant (MDR) H. pylori strains and is upregulated by SpoT. Through further research, we determined that gluP is involved in H. pylori biofilm formation and multidrug resistance. Furthermore, the average expression level of gluP in the clinical MDR strains (C-MDR) was considerably higher than that in the clinical drug-sensitive strains (C-DSS). Taken together, our results revealed a novel molecular mechanism of H. pylori resistance to multidrug exposure.

MiRNA-491-5p inhibits cell proliferation, invasion and migration via targeting JMJD2B and serves as a potential biomarker in gastric cancer.

Our previous work discovered that the histone demethylase JMJD2B (KDM4B) plays oncogenic roles in gastric carcinogenesis, but the regulatory mechanism of JMJD2B in gastric cancer has not been well defined. It has been revealed that microRNAs function as gene regulators by binding to the 3'UTR of mRNAs to inhibit gene expression. In this study, we found that miR-491-5p suppressed cell proliferation, invasion and migration by directly targeting the JMJD2B 3'UTR in gastric cancer. Moreover, miR-491-5p was decreased in GC tissues compared with adjacent normal tissues, and JMJD2B had the inverse expression pattern. In contrast to healthy individuals, GC patients had lower miR-491-5p expression in serum (P<0.0001). Our data indicate that miR-491-5p serves as a tumor suppressor in GC and might be a novel potential biomarker for the detection of gastric cancer.

Combined optimization of N-terminal site-specific PEGylation of recombinant hirudin using response surface methodology and kinetic analysis.

In this study, a combined optimization method was developed to optimize the N-terminal site-specific PEGylation of recombinant hirudin variant-2 (HV2) with different molecular weight mPEG-propionaldehyde (mPEG-ALD), which is a multifactor-influencing process. The HV2-PEGylation with 5 kDa mPEG-ALD was first chosen to screen significant factors and determine the locally optimized conditions for maximizing the yield of mono-PEGylated product using combined statistical methods, including the Plackett-Burman design, steepest ascent path analysis, and central composition design for the response surface methodology (RSM). Under the locally optimized conditions, PEGylation kinetics of HV2 with 5, 10, and 20 kDa mPEG-ALD were further investigated. The molar ratio of polyethylene glycol to HV2 and reaction time (the two most significant factors influencing the PEGylation efficiency) were globally optimized in a wide range using kinetic analysis. The data predicted by the combined optimization method using RSM and kinetic analysis were in good agreement with the corresponding experiment data. PEGylation site analysis revealed that almost 100% of the obtained mono-PEGylated-HV2 was modified at the N-terminus of HV2. This study demonstrated that the developed method is a useful tool for the optimization of the N-terminal site-specific PEGylation process to obtain a homogeneous mono-PEGylated protein with desirable yield.

In vitro and in vivo effects of suloctidil on growth and biofilm formation of the opportunistic fungus Candida albicans.

As the most frequent fungal pathogen in humans, Candida albicans can develop serious drug resistance because its biofilms are resistant to most antifungal agents; this leads to an urgent need to develop novel antifungals. Here, we evaluated the efficacy of an antithrombotic drug, suloctidil, against C. albicans biofilms in vitro and in vivo. We found that suloctidil is effective to inhibit C. albicans biofilm, with a minimum inhibitory concentration (MIC80) of 4 µg/mL, a biofilm inhibiting concentration (BIC80) of 16 µg/mL and a biofilm eradicating concentration (BEC80) of 64 µg/mL. Furthermore, the concentration-dependent characteristics of suloctidil were shown by its time-kill curves. Scanning electron microscopy images clearly revealed the morphological effects of suloctidil on biofilm. Yeast-to-hyphal form switching is a key virulence factor of C. albicans; therefore, we performed hyphal growth tests and observed that suloctidil inhibited yeast-to-hyphal form switching. This result was consistent with the down-regulation of hypha-specific gene (HWP1, ALS3, and ECE1) expression levels after suloctidil treatment. In vivo, 256 µg/mL of suloctidil significantly reduced fungal counts (P<0.01) compared to that in groups without treatment; the treatment group induced a slight histological reaction, especially when the treatment lasted for 5 days (P<0.01). Taken together, our data suggest that suloctidil is a potential antifungal agent.

Metformin Inhibits Porphyromonas gingivalis Lipopolysaccharide-Influenced Inflammatory Response in Human Gingival Fibroblasts via Regulating Activating Transcription Factor-3 Expression.

BACKGROUND: Chronic periodontitis, one of the most prevalent oral diseases, is associated with Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS) infection and has profound effects on type 2 diabetes mellitus (t2DM). Metformin, a well-known antidiabetic agent, has been reported to exert anti-inflammatory effects on various cells. This study aims to investigate the role of metformin on LPS-influenced inflammatory response in human gingival fibroblasts (HGFs). METHODS: Dose-dependent additive effects of metformin on LPS-influenced HGFs were detected. Cell-counting assay was used to determine effects of metformin and LPS on viability of HGFs. Enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction (qRT-PCR) were applied to detect levels of interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α in differently treated cells. Activating transcription factor-3 (ATF3) small interfering (si)RNA transfection was used to determine the mechanism of metformin action, and the transfection efficiency was observed by fluorescence microscope. Effects of ATF3 knockdown were determined by qRT-PCR and Western blot. RESULTS: Results showed that 5 µg/mL Pg LPS and 0.1, 0.5, and 1 mM metformin exhibited no toxicity to HGFs, and metformin inhibited LPS-influenced IL-1ß, IL-6, and TNF-α production in a dose-dependent manner. Metformin and LPS could synergistically facilitate ATF3 expression, and ATF3 knockdown abolished inhibitory effects of metformin on LPS-influenced inflammatory cytokine production in HGFs. CONCLUSION: The present study confirms that metformin suppresses LPS-enhanced IL-6, IL-1ß, and TNF-α production in HGFs via increasing ATF3 expression.