Inhibition of ALKBH5 inhibits inflammation and excessive proliferation by promoting TRIM13 m6A modifications in glomerular mesangial cells.

Chronic glomerulonephritis (CGN) refers to the inflammation of glomeruli in the kidneys. Glomerular mesangial cells (GMCs) play a pivotal role in the development of CGN. In the present study, we investigated the impact of ALKBH5, a m6A demethylase, on inflammation and hyperproliferation in mouse glomerular mesangial cells (MMCs) and elucidated the molecular mechanisms contributing to CGN. Western blotting and reverse transcriptase-polymerase chain reaction (RT-qPCR) were employed to evaluate the expression of ALKBH5 and TRIM13. In addition, enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of inflammatory factors (IL-1ß, TNF-α, and IL-10) in the lipopolysaccharide (LPS)-induced MMCs supernatant. Methylated RNA immunoprecipitation (MeRIP) was performed to investigate the effect of ALKBH5 on the levels of TRIM13-m6A mRNA. The stability of TRIM13 mRNA was evaluated using an actinomycin D assay. Significantly elevated expression of ALKBH5 was found in LPS-induced MMCs. Interference with ALKBH5 expression inhibited inflammation and excessive proliferation in LPS-induced MMCs. Moreover, interfering with ALKBH5 expression significantly reduced the levels of TRIM13-m6A modification. The overexpression of TRIM13 in MMCs reversed the inflammation and proliferation induced by ALKBH5 interference. In addition, interference with TRIM13 expression inhibited the activation of the NF-κB pathway and suppressed inflammation and proliferation in MMCs. Inhibiting ALKBH5 hinders inflammation and hyperproliferation by improving TRIM13-m6A modification in glomerular MCs. We believe these findings will further provide insights into the molecular mechanisms and potential therapeutic targets for CGN.

METTL3 modification of circStk4 affects mouse glomerular messangial cell autophagy, proliferation and apotosis by regulating miR-133a-3p/C1 axis.

OBJECTIVE: The study aimed to explore the impact of N6-methyladenosine (m6A) modification in circStk4 on glomerular mesangial cells (GMCs) autophagy, proliferation and apoptosis. METHODS: The interactions between circStk4 and miR-133a-3p, miR-133a-3p and C1 were demonstrated through luciferase reporter assays. The circStk4 localization was analyzed using fluorescence in situ hybridization and nuclear/cytosol fractionation assays. Colorimetric assays, MeRIP-qPCR, and western blot (WB) were employed to confirm the m6A modification of circStk4 and identify the key methylation enzyme. RT-qPCR was conducted to determine the impact of METTL3 on the circStk4 RNA expression. Additionally, CCK-8, flow cytometry, transmission electron microscopy, immunofluorescence, WB and RT-qPCR were employed to investigate the effects of METTL3 or circStk4 on the proliferation, autophagy and apoptosis of GMCs. Enzyme-linked immunosorbent assay was utilized to assess the inflammatory factors. RESULTS: m6A modifications were found in circStk4 and METTL3 was a key methylating enzyme. Furthermore, it was observed that circStk4 competitively bound miR-133a-3p and increased C1 levels. Silencing circStk4 resulted in decreased GMCs proliferation, increased autophagy and apoptosis, and reduced inflammation levels. Additionally, METTL3 played a role in inhibiting GMCs proliferation and promoting autophagy and apoptosis by regulating the circStk4 expression. On verifying the interplay between autophagy, proliferation and apoptosis, and found that the inhibition of autophagy led to an increase in cell proliferation and a decrease in apoptosis. CONCLUSION: m6A modification of circStk4 mediated by METTL3 influenced circStk4 expression and impacted autophagy, proliferation and apoptosis in GMCs via the miR-133a-3p/C1 axis. This discovery introduces a novel therapeutic approach for CGN treatment.

Overexpression of FTO inhibits excessive proliferation and promotes the apoptosis of human glomerular mesangial cells by alleviating FOXO6 m6A modification via YTHDF3-dependent mechanisms.

Background: N6-methyladenosine (m6A) is a prevalent post-transcriptional modification presented in messenger RNA (mRNA) of eukaryotic organisms. Chronic glomerulonephritis (CGN) is characterised by excessive proliferation and insufficient apoptosis of human glomerular mesangial cells (HGMCs) but its underlying pathogenesis remains undefined. Moreover, the role of m6A in CGN is poorly understood. Methods: The total level of m6A modification was detected using the m6A quantification assay (Colorimetric). Cell proliferation was assessed by EdU cell proliferation assay, and cell apoptosis was detected by flow cytometry. RNA sequencing was performed to screen the downstream target of fat mass and obesity-associated protein (FTO). MeRIP-qPCR was conducted to detect the m6A level of forkhead box o6 (FOXO6) in HGMCs. RIP assay was utilized to indicate the targeting relationship between YTH domain family 3 (YTHDF3) and FOXO6. Actinomycin D assay was used to investigate the stability of FOXO6 in HGMCs. Results: The study found that the expression of FTO was significantly reduced in lipopolysaccharide (LPS)-induced HGMCs and renal biopsy samples of patients with CGN. Moreover, FTO overexpression and knockdown could regulate the proliferation and apoptosis of HGMCs. Furthermore, RNA sequencing and cellular experiments revealed FOXO6 as a downstream target of FTO in regulating the proliferation and apoptosis of HGMCs. Mechanistically, FTO overexpression decreases the level of FOXO6 m6A modification and reduces the stability of FOXO6 mRNA in a YTHDF3-dependent manner. Additionally, the decreased expression of FOXO6 inhibits the PI3K/AKT signaling pathway, thereby inhibiting the proliferation and promoting apoptosis of HGMCs. Conclusion: This study offers insights into the mechanism through which FTO regulates the proliferation and apoptosis of HGMCs by mediating m6A modification of FOXO6 mRNA. These findings also suggest FTO as a potential diagnostic marker and therapeutic target for CGN.

Identifying Aging-Related Biomarkers and Immune Infiltration Features in Diabetic Nephropathy Using Integrative Bioinformatics Approaches and Machine-Learning Strategies.

BACKGROUND: Aging plays an essential role in the development of diabetic nephropathy (DN). This study aimed to identify and verify potential aging-related genes associated with DN using bioinformatics analysis. METHODS: To begin with, we combined the datasets from GEO microarrays (GSE104954 and GSE30528) to find the genes that were differentially expressed (DEGs) across samples from DN and healthy patient populations. By overlapping DEGs, weighted co-expression network analysis (WGCNA), and 1357 aging-related genes (ARGs), differentially expressed ARGs (DEARGs) were discovered. We next performed functional analysis to determine DEARGs' possible roles. Moreover, protein-protein interactions were examined using STRING. The hub DEARGs were identified using the CytoHubba, MCODE, and LASSO algorithms. We next used two validation datasets and Receiver Operating Characteristic (ROC) curves to determine the diagnostic significance of the hub DEARGs. RT-qPCR, meanwhile, was used to confirm the hub DEARGs' expression levels in vitro. In addition, we investigated the relationships between immune cells and hub DEARGs. Next, Gene Set Enrichment Analysis (GSEA) was used to identify each biomarker's biological role. The hub DEARGs' subcellular location and cell subpopulations were both identified and predicted using the HPA and COMPARTMENTS databases, respectively. Finally, drug-protein interactions were predicted and validated using STITCH and AutoDock Vina. RESULTS: A total of 57 DEARGs were identified, and functional analysis reveals that they play a major role in inflammatory processes and immunomodulation in DN. In particular, aging and the AGE-RAGE signaling pathway in diabetic complications are significantly enriched. Four hub DEARGs (CCR2, VCAM1, CSF1R, and ITGAM) were further screened using the interaction network, CytoHubba, MCODE, and LASSO algorithms. The results above were further supported by validation sets, ROC curves, and RT-qPCR. According to an evaluation of immune infiltration, DN had significantly more resting mast cells and delta gamma T cells but fewer regulatory T cells and active mast cells. Four DEARGs have statistical correlations with them as well. Further investigation revealed that four DEARGs were implicated in immune cell abnormalities and regulated a wide range of immunological and inflammatory responses. Furthermore, the drug-protein interactions included four possible therapeutic medicines that target four DEARGs, and molecular docking could make this association practical. CONCLUSIONS: This study identified four DEARGs (CCR2, VCAM1, CSF1R, and ITGAM) associated with DN, which might play a key role in the development of DN and could be potential biomarkers in DN.

Identification of Rab7 as an autophagy marker: potential therapeutic approaches and the effect of Qi Teng Xiao Zhuo granule in chronic glomerulonephritis.

CONTEXT: Qi Teng Xiao Zhuo granule (QTXZG) is a traditional Chinese medicine (TCM) used for therapeutic effects on chronic glomerulonephritis (CGN). However, the underlying mechanism remains unclear. OBJECTIVE: To investigate the molecular mechanism of QTXZG on CGN by proteomics. MATERIALS AND METHODS: The CGN model was induced in Sprague-Dawley rats by injecting adriamycin (3.5 mg/kg, Day 1; 3.0 mg/kg, Day 14) twice through the tail vein. Urine samples were collected on the 21st day; and the rats divided randomly into control, adriamycin, QTXZG administration groups. Rats in the QTXZG group received QTXZG (10.8 g/kg); control and adriamycin groups were given physiological saline once per day for 30 days. Proteomics was applied to identify the candidate proteins combined with autophagy database and verified by immunofluorescence (IF) and western blots (WB). RESULTS: 278 differentially expressed proteins (DEPs) were identified based on proteomics and Rab7 was screened as an autophagy protein biomarker. In vitro cell experiments, we found that QTXZG (20%, IC50 = 23.47%) could decrease the expression of NLRP3, Caspase-1, IL-18, IL-1ß, while increasing the expression of Pink1, Parkin, Rab7, Podocalyxin. The cell apoptosis rate increased from 6.68 ± 0.07 to 11.03 ± 0.36%. Overexpression of Rab7 resulted in an increase in autophagy relevant protein expression. DISCUSSION AND CONCLUSION: TCM CGN-regulating herbs (QTXZG) can exert therapeutic effects by affecting the Rab7/Pink1/Parkin pathway to promote mitochondrial autophagy. New breakthroughs in targeted Rab7 may eventually enable such applications.

Astragalus Polysaccharide Ameliorates Renal Inflammatory Responses in a Diabetic Nephropathy by Suppressing the TLR4/NF-κB Pathway.

Background: Diabetic nephropathy (DN), as a chronic inflammatory complication of diabetes, is characterized by hyperglycemia, albuminuria and edema, which ultimately becomes the leading cause of end-stage renal disease (ESRD). Astragalus polysaccharide (APS), extracted from the Astragalus membranaceus, was widely used in the treatment of diabetes mellitus. However, the functional roles of APS ameliorate inflammatory responses in DN, which remain poorly understood. Therefore, the purpose of this study was to explore the molecular mechanism of APS on DN in vivo and vitro models. Methods: We explored the beneficial effects of APS in streptozotocin (STZ)-induced DN rat model and high glucose (HG)-treated glomerular podocyte model. The fasting blood glucose (FBG) and ratio of kidney weight to body weight were measured after 4 weeks of APS treatment. The renal injury parameters containing serum creatinine (Scr), blood urea nitrogen (BUN) and 24 h urinary protein were evaluated. The renal pathological examination was observed by hematoxylin-eosin (HE) staining. The levels of IL-1ß, IL-6 and MCP-1 were evaluated by ELISA assay. The proliferation of podocytes was determined using CCK-8 assay and flow cytometry. qRT-PCR and Western blot analysis were performed to determine the amounts of TLR4/NF-κB-related gene expression. Results: Our results indicated that APS effectively decreased the levels of FBG, BUN, Scr and renal pathological damage when compared with STZ-induced DN model group. Additionally, APS significantly ameliorated renal injury by reducing inflammatory cytokines IL-1ß, IL-6, MCP-1 expression and inhibiting the TLR4/NF-κB pathway activity in DN rats. Consistent with the results in vitro, the HG-induced inflammatory response and proliferation of glomerular podocytes were also alleviated through APS administration. Conclusion: We found that APS ameliorated DN renal injury, and the mechanisms perhaps related to relieving inflammatory responses and attenuating the TLR4/NF-κB signaling pathway.

Construction of chronic glomerulonephritis­related lncRNA­mRNA regulatory network and lncRNA­-miRNA­mRNA ceRNA network by bioinformatics analysis.

Long non-coding RNAs (lncRNAs) are ncRNA transcripts >200 nucleotides that are important genetic regulators. LncRNAs can directly regulate mRNA through a lncRNA-mRNA regulatory mode and can also regulate mRNA through competitive binding to micro (mi)RNA, which is generally known as the competitive endogenous RNA (ceRNA) network. The present study evaluated the functional roles and regulatory networks of lncRNAs in chronic glomerulonephritis (CGN). The proliferative ability of mouse glomerular mesangial cells (GMCs) induced by different concentrations of lipopolysaccharide (LPS) was assessed using the Cell Counting Kit-8 assay, and RNA sequencing (RNA-seq) was performed to identify differentially expressed lncRNAs in LPS-induced GMCs. Based on the sequencing results, six lncRNAs were selected for validation using reverse transcription-quantitative PCR (RT-qPCR). Furthermore, the lncRNA-mRNA regulatory network and the lncRNA-miRNA-mRNA ceRNA network were constructed to assess the role and mechanism of CGN-related lncRNAs. To elucidate the biological functions of lncRNAs, Gene Ontology (GO) biological process term enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed on all mRNAs involved in the lncRNA-mRNA regulatory network and in the ceRNA network. A total of 1,532 differentially expressed lncRNAs, including 594 upregulated lncRNAs and 938 downregulated lncRNAs, were identified using RNA-seq. The results of RT-qPCR validation were consistent with RNA-seq results. An lncRNA-mRNA regulatory network, including 236 lncRNAs and 556 mRNAs, and a ceRNA network, including 6 lncRNAs, 18 miRNAs and 419 mRNAs, were successfully constructed. The GO biological process term enrichment and KEGG pathway enrichment analyses demonstrated that those lncRNAs were often related to inflammatory response and substance metabolism. The present study identified key CGN-related lncRNAs in LPS-induced GMCs, and further demonstrated a global view of the lncRNA-mRNA regulatory network and ceRNA network involved in CGN. These results offered novel insights into the roles of lncRNAs in the pathogenesis of CGN and identified potential diagnostic biomarkers.

RNA sequencing reveals the mechanism of FTO in inhibiting inflammation and excessive proliferation of lipopolysaccharide-induced human glomerular mesangial cells.

Chronic glomerulonephritis (CGN) is a leading cause of end-stage renal disease in China; thus, there is an urgent need for effective therapeutic targets and strategies for CGN treatment. However, studies on CGN pathogenesis are limited. In this study, we found that the fat mass and obesity-associated protein (FTO) was significantly decreased in the lipopolysaccharide (LPS)-induced human glomerular mesangial cells (HGMCs) (P < 0.01) and kidney tissues of CGN patients (P < 0.05). Moreover, double-labeling immunofluorescence and flow cytometry assays demonstrated that the overexpression of FTO could inhibit inflammation and excessive proliferation of HGMCs. Furthermore, RNA-sequencing (RNA-seq) and real-time quantitative polymerase chain reaction (RT-qPCR) analyses revealed that FTO overexpression induced differential expression of 269 genes (absolute fold change ≥ 2 and P-value < 0.05), including 143 upregulated and 126 downregulated genes. Further functional analysis of these differentially expressed genes by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses suggested that FTO possibly mediates its inhibitory function by regulating the mammalian target of rapamycin (mTOR) signaling pathway and substance metabolism. Lastly, analysis of the PPI network and further identification of the top 10 hub genes (RPS15, RPS18, RPL18A, GNB2L1, RPL19, EEF1A1, RPS25, FAU, UBA52, and RPS6) indicated that FTO mediates its function by affecting the ribosomal proteins. Therefore, in this study, we elucidated the important role of FTO in the regulation of inflammation and excessive proliferation of HGMCs, suggesting FTO administration as a suitable therapeutic intervention for CGN.

Comprehensive analysis of m6A modification lncRNAs in high glucose and TNF-α induced human umbilical vein endothelial cells.

N6-methyladenosine (m6A) RNA methylation, as a reversible epigenetic modification of mammalian mRNA, holds a critical role in multiple biological processes. m6A modification in Long non-coding RNAs (lncRNAs) has increasingly attracted more attention in recent years, especially in diabetics, with or without metabolic syndrome. We investigated via m6A-sequencing and RNA-sequencing the differentially expressed m6A modification lncRNAs by high glucose and TNF-α induced endothelial cell dysfunction in human umbilical vein endothelial cells. Additionally, gene ontology and kyoto encyclopedia of genes and genomes analyses were performed to analyze the biological functions and pathways for the target of mRNAs. Lastly, a competing endogenous RNA network was established to further reveal a regulatory relationship between lncRNAs, miRNAs and mRNAs. A total of 754 differentially m6A-methylated lncRNAs were identified, including 168 up-regulated lncRNAs and 266 down-regulated lncRNAs. Then, 119 significantly different lncRNAs were screened out, of which 60 hypermethylated lncRNAs and 59 hypomethylated lncRNAs. Moreover, 122 differentially expressed lncRNAs were filtered, containing 14 up-regulated mRNAs and 18 down-regulated lncRNAs. Gene ontology and kyoto encyclopedia of genes and genomes analyses analyses revealed these targets were mainly associated with metabolic process, HIF-1 signaling pathway, and other biological processes. The competing endogenous RNA network revealed the regulatory relationship between lncRNAs, miRNAs and mRNAs, providing potential targets for the treatment and prevention of diabetic endothelial cell dysfunction. This comprehensive analysis for lncRNAs m6A modification in high glucose and TNF-α-induced human umbilical vein endothelial cells not only demonstrated the understanding of characteristics of endothelial cell dysfunction, but also provided the new targets for the clinical treatment of diabetes. Private information from individuals will not be published. This systematic review also does not involve endangering participant rights. Ethical approval will not be required. The results may be published in a peer-reviewed journal or disseminated at relevant conferences.

Metabolomic Analysis of Stephania tetrandra-Astragalus membranaceus Herbal Pair-Improving Nephrotic Syndrome Identifies Activation of IL-13/STAT6 Signaling Pathway.

The Stephania tetrandra−Astragalus membranaceus herbal pair (FH) is a classic herbal pair widely used in the treatment of nephrotic syndrome (NS). The effects of Stephania tetrandra (FJ) and Astragalus membranaceus (HQ) on NS have been reported, but the mechanism of their combination on the improvement of NS are still unclear. The NS model was established by injecting adriamycin into the tail vein. FH intervention reduced the levels of serum triglyceride, total cholesterol, interleukin-6 (IL-6), blood urea nitrogen (BUN), urinary protein, and the gene expression levels of aquaporin 2 (AQP2) and arginine vasopressin (AVP) in NS rats. In addition, FH improved kidney injury in NS rats by inhibiting the expression of interleukin 13 (IL-13), phospho-signal transducers, and activators of transcription 6 (p-STAT6), Bax, cleaved-caspase3, while promoting the expression of Bcl-2. By comprehensive comparison of multiple indexes, the effects of FH on lipid metabolism, glomerular filtration rate, and inflammation were superior to that of FJ and HQ. Metabonomic studies showed that, compared with FJ and HQ, FH intervention significantly regulated tricarboxylic acid (TCA) cycle, cysteine and methionine metabolism, and alanine, aspartic acid and glutamic acid metabolism. Pearson correlation analysis showed that succinic acid and L-aspartic acid were negatively correlated with urinary protein, cystatin C (Cys C) and BUN (p < 0.05). In summary, FH could reduce renal injury and improve NS through inhibiting the IL-13/STAT6 signal pathway, regulating endogenous metabolic pathways, such as TCA cycle, and inhibiting the expression of AQP2 and AVP genes. This study provides a comprehensive strategy to reveal the mechanism of FH on the treatment of NS, and also provides a reasonable way to clarify the compatibility of traditional Chinese medicine.

High-throughput data on circular RNA reveal novel insights into chronic glomerulonephritis.

BACKGROUND: Circular RNAs (circRNAs), a unique novel type of RNA, have been widely reported to be involved in physiologic and pathologic processes in humans. However, the exact molecular pathogenesis of circRNAs in chronic glomerulonephritis (CGN) is far from clear. OBJECTIVE: This paper aims to evaluate the specific expression profile of circRNAs in renal cortex tissues from Adriamycin-induced CGN rats. METHODS: CircRNAs were screened in renal cortex tissues from 3 CGN rats and 3 control rats by using high-throughput sequencing (HTS). Then, 4 circRNAs were selected randomly for verification by quantitative real-time polymerase chain reaction (qRT-PCR). In addition, the differentially expressed (DE) circRNAs were analyzed by bioinformatics methods. RESULTS: In total, 31 significantly DE circRNAs were identified, which revealed their potential roles in CGN; in particular, we found that 4 confirmed altered circRNAs (rno-circ-RNAs 689, 3217, 1327, and 5001) might play important roles in the development of CGN. CONCLUSION: This study reveals a cluster of circRNAs that are DE in Adriamycin-induced CGN rats, which brings us closer to understanding the pathogenic mechanisms and may provide new potential targets for clinical treatment.

Bioinformatics Analysis of the Mechanisms of Diabetic Nephropathy via Novel Biomarkers and Competing Endogenous RNA Network.

Diabetic nephropathy (DN) is one of the common chronic complications of diabetes with unclear molecular mechanisms, which is associated with end-stage renal disease (ESRD) and chronic kidney disease (CKD). Our study intended to construct a competing endogenous RNA (ceRNA) network via bioinformatics analysis to determine the potential molecular mechanisms of DN pathogenesis. The microarray datasets (GSE30122 and GSE30529) were downloaded from the Gene Expression Omnibus database to find differentially expressed genes (DEGs). GSE51674 and GSE155188 datasets were used to identified the differentially expressed microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), respectively. The DEGs between normal and DN renal tissues were performed using the Linear Models for Microarray (limma) package. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to reveal the mechanisms of DEGs in the progression of DN. The protein-protein interactions (PPI) of DEGs were carried out by STRING database. The lncRNA-miRNA-messenger RNA (mRNA) ceRNA network was constructed and visualized via Cytoscape on the basis of the interaction generated through the miRDB and TargetScan databases. A total of 94 significantly upregulated and 14 downregulated mRNAs, 31 upregulated and 121 downregulated miRNAs, and nine upregulated and 81 downregulated lncRNAs were identified. GO and KEGG pathways enriched in several functions and expression pathways, such as inflammatory response, immune response, identical protein binding, nuclear factor kappa b (NF-κB) signaling pathway, and PI3K-Akt signaling pathway. Based on the analysis of the ceRNA network, five differentially expressed lncRNAs (DElncRNAs) (SNHG6, KCNMB2-AS1, LINC00520, DANCR, and PCAT6), five DEmiRNAs (miR-130b-5p, miR-326, miR-374a-3p, miR-577, and miR-944), and five DEmRNAs (PTPRC, CD53, IRF8, IL10RA, and LAPTM5) were demonstrated to be related to the pathogenesis of DN. The hub genes were validated by using receiver operating characteristic curve (ROC) and real-time PCR (RT-PCR). Our research identified hub genes related to the potential mechanism of DN and provided new lncRNA-miRNA-mRNA ceRNA network that contributed to diagnostic and potential therapeutic targets for DN.

Long Non-Coding NONRATG001910.2 Promotes the Proliferation of Rat Mesangial Cell Line HBZY-1 Through the miR-339-3p/CTNNB1 Axis.

Chronic glomerulonephritis (CGN) is one of the leading causes of end-stage renal disease (ESRD). A growing body of literature emphasizes the important role of long non-coding RNAs (lncRNAs) in the development and progression of the disease. However, the function of NONRATG001910.2 in the development of CGN was not well understood. This research aimed to investigate the effect of NONRATG001910.2 on CGN and revealed its potential molecular mechanisms. In this work, the expression of NONRATG001910.2 was detected by quantitative real-time polymerase chain reaction (qRT-RCR) in cell lines. We found that NONRATG001910.2 was significantly up-regulated in lipopolysaccharide (LPS) induced cells. High NONRATG001910.2 levels were associated with the development of CGN. In addition, NONRATG001910.2 knockdown inhibited cell proliferation and cell cycle. At the same time, we found that up-regulation of microRNA-339-3p (miR-339-3p) abrogated the biological roles of NONRATG001910.2 up-regulation. Moreover, the knockdown of CTNNB1 can upregulate miR-339-3p expression, thereby inhibiting cell proliferation. In conclusion, these results demonstrated that NONRATG001910.2 in LPS-stimulated rat mesangial cell line HBZY-1 (HBZY-1) by targeting miR-339-3p, which subsequently promotes the expression of CTNNB1, and suggested that NONRATG001910.2 may be a potential biomarker.

Salvianolic acid B acts against non­small cell lung cancer A549 cells via inactivation of the MAPK and Smad2/3 signaling pathways.

Salvianolic acid B (Sal B) is a potential cytotoxic polyphenol against cancer. In the present study the effect of Sal B and its molecular mechanism were investigated in the non­small cell lung cancer (NSCLC) A549 cell line. The TGF­ß/MAPK/Smad signaling axis was explored. A549 cells were co­cultured with and without different concentrations of Sal B (25, 50 and 100 µM respectively) and TGF­ß1 (9 pM) for 24 h. Cell epithelial­mesenchymal transition (EMT), cell migration, cell cycle distribution, autophagy and apoptosis were assessed by western blotting (WB), wound healing assay and flow cytometry, respectively. Moreover, activation of MAPK, Smad2/3 and the downstream target, plasminogen activator inhibitor 1 (PAI­1), were assessed by WB. The results demonstrated that Sal B inhibited TGF­ß1­induced EMT and migration of A549 cells, hampered cell cycle progression and induced cell autophagy and apoptosis. Furthermore, Sal B inactivated MAPK signaling pathways and the phosphorylation of Smad2/3, especially the phosphorylation of Smad3 at the linker region, which resulted in decreased protein expression levels of PAI­1 in TGF­ß1­stimulated A549 cells. Overall, these results demonstrated that Sal B may have a potential therapeutic effect against NSCLC in vitro. The results of the present study indicated that the underlying active mechanism of Sal B in NSCLC may be closely related to the impeded activation of the MAPK and Smad2/3 signaling pathways. Therefore, Sal B may be a potential candidate NSCLC therapeutic agent.

Network pharmacology-based identification of miRNA expression of Astragalus membranaceus in the treatment of diabetic nephropathy.

ABSTRACT: Diabetic nephropathy (DN) is a common microvascular complication of diabetic patients, along with hypertension, hyperlipemia, proteinuria, edema, and other clinical manifestations. Astragalus membranaceus (AM) is a traditional Chinese medicine and has shown significant clinical efficacy against DN. However, the overall molecular mechanism of this therapeutic effect has not been entirely elucidated. Using network pharmacology, we aimed to identify the key active ingredients and potential pharmacological mechanisms of AM in treating DN and provide scientific evidence of its clinical efficacy.The active ingredients of AM were obtained from the traditional Chinese medicine systems pharmacology database, and the potential targets of AM were identified using the therapeutic target database. DN-related target genes were acquired from the Gene Expression Omnibus microarray dataset GSE1009 and 3 widely used databases-DisGeNET, GeneCards, and Comparative Toxicogenomics Database. The DN-AM common target protein interaction network was established by using the STRING database. Active ingredients candidate targets proteins networks were constructed using Cytoscape software for visualization. Additionally, gene ontology (GO) and Kyoto encyclopedia of genes and genomes pathway analyses were performed using the Database for Annotation, Visualization, and Integrated Discovery database. Target-regulating microRNAs (miRNAs) of these hub genes were obtained from the therapeutic target database, which could then be used for further identification of AM-regulated key miRNAs.A total of 17 active ingredients and 214 target proteins were screened from AM. 61 candidate co-expressed genes with therapeutic effects against DN were obtained and considered as potential therapeutic targets. GO and Kyoto encyclopedia of genes and genomes enrichment analysis showed that these genes were mainly involved in inflammatory response, angiogenesis, oxidative stress reaction, HIF signaling pathway, tumor necrosis factor signaling pathway, and VEGF signaling pathway. In all, 636 differentially expressed genes were identified between the DN patients and control group by using microarray data, GSE1009. Lastly, VEGFA, epidermal growth factor receptor, STAT1, and GJA1 were screened as hub genes. The relationships between miRNAs and hub genes were constructed, which showed that miR-302-3p, miR-372-3p, miR-373-3p, and miR-520-3p were regulated by VEGFA and epidermal growth factor receptor. Meanwhile, VEGFA also influenced miR-15-5p, miR-16-5p, miR-17-5p, miR-20-5p, miR-93-5p, miR-106-5p, miR-195-5p, miR-424-5p, miR-497-5p, and miR-519-3p. In addition, miR-1-3p and miR-206 were regulated by VEGFA and GJA1, and miR-23-3p was regulated by STAT1 and GJA1.To our knowledge, this study revealed for the first time the characteristic multiple components, multiple targets, and multiple pathways of AM that seem to be the underlying mechanisms of action of AM in the treatment of DN with respect to miRNAs.Private information from individuals will not be published. This systematic review also does not involve endangering participant rights. Ethical approval will not be required. The results may be published in a peer-reviewed journal or disseminated at relevant conferences.

[miR-324-5p inhibits lipopolysaccharide-induced proliferation of rat glomerular mesangial cells by regulating the Syk/Ras/c-fos pathway].

OBJECTIVE: To investigate the effect of miR-324-5p on the proliferation of rat glomerular mesangial (HBZY-1) cells and the role of Syk/Ras/c-fos signaling pathway in mediating this effect. METHODS: HBZY-1 cells cultured in vitro were transiently transfected with miR-324-5p mimics or miR-324-5p-mimics-NC followed by treatment with lipopolysaccharide (LPS). MTT assay was used to detect the proliferation activity of HBZY-1 cells, and RT-qPCR was used to detect the expressions of miR-324-5p and the mRNA expressions of Syk, Ras, MEK1/2, ERK1/2 and c-fos mRNA. The protein expressions of p-Syk, Ras, p-MEK1/2, p-ERK1/2 and c-Fos were detected by Western blotting and immunofluorescence assay. RESULTS: MTT assay showed that exposure to LPS significantly enhanced the proliferative activity of HBZY-1 cells. Compared with the cells treated with LPS and LPS + mimics NC, the cells transfected with miR-324-5p mimics prior to LPS exposure exhibited significantly lowered proliferative activity. Transfection with miR-324-5p mimics significantly lowered the mRNA expressions of Syk, Ras, MEK1/2, ERK1/2 and c-fos and the protein expressions of p-Syk, Ras, MEK1/2, ERK1/2 and c-Fos (P < 0.05), and reduced numbers of cells positive for p-Syk, Ras, p-MEK1/2, p-ERK1/2 and c-Fos proteins following LPS exposure. CONCLUSIONS: miR-324-5p can inhibit the proliferation of rat chronic glomerulonephritis cells induced by LPS by inhibiting Syk/Ras/c-fos signaling pathway and may potentially serve as a diagnostic indicator and a therapeutic target for chronic glomerulonephritis.

[Identification of chemical constituents in Huangdi Anxiao Capsules by UPLC-Q-TOF-MS~E combined with UNIFI software].

In order to clarify the main chemical constituents of Huangdi Anxiao Capsules, an ultra-high performance liquid coupled with quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS~E) combined with Waters UNIFI software were successfully used to rapidly identify the chemical constituents in Huangdi Anxiao Capsules. The mass spectrometry data of chemical constituents from Huangdi Anxiao Capsules were collected by UPLC-Q-TOF-MS~E, and their structures were identified by the results of UNIFI software according to relative retention time of reference standards, MS feature fragments and literature data of each compound. A total of 100 compounds in Huangdi Anxiao Capsules were identified, including 25 compounds from Pueraria Lobate Radix, 22 compounds from Coptis Rhizoma, 6 compounds from Ophiopogonis Radix, 14 compounds from Eriobotryae Folium, 22 compounds from Rehmanniae Radix, and 15 compounds from Notoginseng Radix et Rhizoma. Among them, 3 compounds were common components. These 100 compounds included flavonoids, alkaloids, saponins and organic acids. This study systematically analyzed the chemical composition of Huangdi Anxiao Capsules, so as to provide evidences for defining the chemical material basis of Huangdi Anxiao Capsules.

Uncovering the Mechanism of Astragalus membranaceus in the Treatment of Diabetic Nephropathy Based on Network Pharmacology.

BACKGROUND: Diabetic nephropathy (DN), characterized by hyperglycemia, hypertension, proteinuria, and edema, is a unique microvascular complication of diabetes. Traditional Chinese medicine (TCM) Astragalus membranaceus (AM) has been widely used for DN in China while the pharmacological mechanisms are still unclear. This work is aimed at undertaking a network pharmacology analysis to reveal the mechanism of the effects of AM in DN. Materials and Methods. In this study, chemical constituents of AM were obtained via Traditional Chinese Medicine Systems Pharmacology Database (TCMSP), and the potential targets of AM were identified using the Therapeutic Target Database (TTD). DisGeNET and GeneCards databases were used to collect DN-related target genes. DN-AM common target protein interaction network was established by using the STRING database. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were carried out to further explore the DN mechanism and therapeutic effect of AM. The network diagrams of the active component-action target and protein-protein interaction (PPI) networks were constructed using Cytoscape software. RESULTS: A total of 16 active ingredients contained and 78 putative identified target genes were screened from AM, of which 42 overlapped with the targets of DN and were considered potential therapeutic targets. The analysis of the network results showed that the AM activity of component quercetin, formononetin, calycosin, 7-O-methylisomucronulatol, and quercetin have a good binding activity with top ten screened targets, such as VEGFA, TNF, IL-6, MAPK, CCL3, NOS3, PTGS2, IL-1ß, JUN, and EGFR. GO and KEGG analyses revealed that these targets were associated with inflammatory response, angiogenesis, oxidative stress reaction, rheumatoid arthritis, and other biological process. CONCLUSIONS: This study demonstrated the multicomponent, multitarget, and multichannel characteristics of AM, which provided a novel approach for further research of the mechanism of AM in the treatment of DN.

Effect of the traditional Chinese medicine Qi Teng Xiao Zhuo granules on chronic glomerulonephritis rats studied by using long noncoding RNAs expression profiling.

AIM OF THE STUDY: Chronic glomerulonephritis (CGN) is the most common form of primary glomerular disease. Qi Teng Xiao Zhuo granules have been proposed as a prescription of traditional Chinese medicine (TCM) for treatment of CGN, however,the comprehensive molecular mechanism underlying this therapeutic effectremains unclear to date. Our study aimed to evaluate and analyze the possible roles and molecular mechanisms of Qi Teng Xiao Zhuo granule-mediated treatment of CGN induced by adriamycin in rats. MATERIALS AND METHODS: RNA-sequencing and real-time polymerase chain reaction (RT-PCR) were applied to identify specifically expressed long noncoding RNAs (lncRNAs) in glomerular tissues of rats from the control group, adriamycin-induced group, and Qi Teng Xiao Zhuo granules group (n = 3). Differentially expressed lncRNAs and mRNAs (messengerRNAs) were screened out among the 3 groups. Gene ontology (GO) and pathway enrichment analyses were performed to analyze the biological functions and pathways for mRNAs. LncRNA-mRNA co-expression network was constructed to analyse for the genes. The protein-protein interaction (PPI) network was visualized. RESULTS: A total of 473 significantly up and down-regulated lncRNAs, 753 up and down-regulated mRNAs were identified. Additionally, it is worth noting that TOP2a (topoisomerase (DNA) II alpha), with the highest connectivity degree in PPI network, was enriched in variouskinds of pathways. Coding-non-coding gene co-expression networks (CNC network) were drawn based on the correlation analysis between lncRNAs and mRNAs. Ten lncRNAs, NONRATT009275.2, NONRATT025409.2, NONRATT025419.2, MSTRG.7681.1, ENSRNOT00000084373, NONRATT000512.2, NONRATT006734.2, ENSRNOT00000084386, NONRATT021738.2, ENSRNOT00000084080, were selected to analyse the relationship between LncRNAs and Qi Teng Xiao Zhuo granules via the CNC network (Coding-non-coding gene co-expression networks) and GO analysis. Real-time PCR results confirmed that the six lncRNAs were specifically expressed in the Qi Teng Xiao Zhuo granules rats. CONCLUSIONS: The ten lncRNAs might play important roles in the Qi Teng Xiao Zhuo granules treatment of CGN. Key genes, such as Ptprc (protein tyrosine phosphatase, receptor type, C), TOP2a, Fos (FBJ osteosarcoma oncogene), Myc (myelocytomatosis oncogene), etc, may be crucial biomarkers for Qi Teng Xiao Zhuo granules.

A rotifer-derived paralytic compound prevents transmission of schistosomiasis to a mammalian host.

Schistosomes are parasitic flatworms that infect over 200 million people, causing the neglected tropical disease, schistosomiasis. A single drug, praziquantel, is used to treat schistosome infection. Limitations in mass drug administration programs and the emergence of schistosomiasis in nontropical areas indicate the need for new strategies to prevent infection. It has been known for several decades that rotifers colonizing the schistosome's snail intermediate host produce a water-soluble factor that paralyzes cercariae, the life cycle stage infecting humans. In spite of its potential for preventing infection, the nature of this factor has remained obscure. Here, we report the purification and chemical characterization of Schistosome Paralysis Factor (SPF), a novel tetracyclic alkaloid produced by the rotifer Rotaria rotatoria. We show that this compound paralyzes schistosome cercariae and prevents infection and does so more effectively than analogous compounds. This molecule provides new directions for understanding cercariae motility and new strategies for preventing schistosome infection.