The potential role of N6-methyladenosine modification of LncRNAs in contributing to the pathogenesis of chronic glomerulonephritis.

BACKGROUND: Increasing evidence indicates that N6-methyladenosine (m6A) modification of mRNAs has been shown to play a critical role in the occurrence and development of many diseases, while little is known about m6A modification in long non-coding RNAs (LncRNAs). Our study aims to investigate the potential functions of LncRNA m6A modifications in lipopolysaccharide (LPS)-induced mouse mesangial cells (MMCs), providing us with a new perspective on the molecular mechanisms of chronic glomerulonephritis (CGN) pathogenesis. METHODS: Differentially methylated LncRNAs were identified by Methylated RNA immunoprecipitation sequencing (MeRIP-seq). LncRNA-mRNA and LncRNA-associated LncRNA-miRNA-mRNA (CeRNA) networks were constructed by bioinformatics analysis. Furthermore, we utilized gene ontology (GO) and pathway enrichment analyses (KEGG) to explore target genes from co-expression networks. In addition, the total level of m6A RNA methylation and expression of methyltransferase and pro-inflammatory cytokines were detected by the colorimetric quantification method and western blot, respectively. Cell viability and cell cycle stage were detected by cell counting kit-8 (CCK-8) and flow cytometry. RESULTS: In total, 1141 differentially m6A-methylated LncRNAs, including 529 hypermethylated LncRNAs and 612 hypomethylated LncRNAs, were determined by MeRIP-seq. The results of GO and KEGG analysis revealed that the target mRNAs were mainly enriched in signal pathways, such as the NF-kappa B signaling pathway, MAPK signaling pathway, Toll-like receptor signaling pathway, and apoptosis signaling pathway. In addition, higher METTL3 expression was found in CGN kidney tissues using the GEO database. METTL3 knockdown in MMC cells drastically reduced the levels of m6A RNA methylation, pro-inflammatory cytokines IL6 and TNF-α, and inhibited cell proliferation and cycle progression. CONCLUSIONS: Our findings provide a basis and novel insight for further investigations of m6A modifications in LncRNAs for the pathogenesis of CGN.

[Effect of Huangdi Anxiao Capsules on zebrafish vascular lesions induced by high glucose and high fat].

Zebrafish of different strains with 5 dpf (5 days post-fertilization) were selected and fed with 0.2% high-fat diet for 8 h and 3% glucose solution for 16 halternatively during the day and night for 4 consecutive days. The zebrafish model was established and randomly divided into model group, Huangdi Anxiao Capsules (260 mg·L⁻¹) group and pioglitazone (32 mg·L⁻¹) group. The drug treatment groups were given the water-soluble drugs, with a volume of 25 mL, and incubated in a 28 °C incubator for 4 days. To detect the exposure to the corresponding drugs, the normal control group was set up. Thirty zebrafish were included in each group. The effect of Huangdi Anxiao Capsules on vascular wall thickness, fluorescence intensity of islet beta cells, fluorescence intensity of macrophages, and blood flow velocity of zebrafish were detected. The expressions of vascular endothelial growth factor (vegfaa) and angiotensin converting enzyme (ACE) were detected by RT-PCR. The results showed that compared with the model group, Huangdi Anxiao Capsules can significantly reduce the thickness of the blood vessel wall, increase the fluorescence intensity of islet ß cells and macrophages, increase the blood flow velocity in vivo, and decrease the ACE and vegfaa expressions in zebrafish. It is suggested that Huangdi Anxiao Capsules may alleviate zebrafish vascular lesions by regulating the expressions of ACE and vegfaa.

[Determination and pharmacokinetics of main components for Psoralea corylifolia-Myristica fragrants drug pair by using UPLC-MS/MS].

To conduct multiple-reaction monitoring(MRM) quantitative analysis with ultra-high performance liquid chromatography coupled with mass spectrometry method(UPLC-MS/MS), determine the concentrations of psoralen, isopsoralen, bakuchiol and dehydrodiisoeugenol in plasma under positive iron mode with chloramghenicol as internal standard, and investigate the pharmacokinetics process of the main components before and after oral administration of drug pair Psoralea corylifolia -Myristica fragrants. Thirty-six SD rats were randomly divided into three group(A, B, C) and received P. corylifolia extract, P. corylifolia-M. fragrants extract, and M. fragrants extract respectively by intragastric administration. The plasma samples were collected at different time points. In the plasma samples, psoralen, isopsoralen, bakuchiol and dehydrodiisoeugenol showed good linear relationship within concentration rages of 0.098 125 to 39.25, 0.084 37 to 33.75, 0.046 875 to 18.75, and 0.11 to 2.2 mg•L⁻¹ respectively. The precision and stability results showed that the determination method of plasma concentration for such compositions was stable and reliable. The pharmacokinetic parameters obtained by DAS 2.0 showed varying differences before and after compatibility. According to the experimental results, the compatibility of P. corylifolia and M. fragrants can significantly impact the pharmacokinetic process of main components, expand their distribution and accelerate their metabolism and elimination in vivo.

[Simultaneous Determination of Seven Chemical Markers and Preliminary Identification of Chemical Constituents in Daodi Psoraleae Fructus-Myristicae Semen Chinese Drug Pair].

OBJECTIVE: To study the simultaneous determination method of daodi Psoraleae Fructus-Myristicae Semen Chinese drug pair for the seven ingredients, and Psoraleae Fructus-Myristicae Semen Chinese drug pair on the chemical composition of initial ownership and identification. METHODS: UPLC BEH C18 column (2.1 mm x 100 mm, 1.7 µm) was used in the determination. The flow rate was kept at 0.25 mL/min, and 2 µL of standard and sample solution were injected in each run. The mobile phase was consisted of acetonitrile and water using a gradient elution. The UPLC/Q-TOF MS condition: Waters HSS T3 (100 mm x 2.1 mm,1.7 µm); capillary voltage 3.0 kV (positive ion mode) and 2.5 kV (negative ion mode); Mass spectrometric detection was carried out on a Waters Xevo G2 Q/ TOF mass spectrometer equipped with an ESI source operating in both positive and negative ion modes. The parameters of the mass spectrometer under the ESI mode were as follows: ion source temperature 110 °C, cone gas flow 50 L/h, desolvation gas temperature 450 °C, desolvation gas flow 800 L/h. RESULTS: The seven chemical markers in the selected linear range had good linearity. The recoveries were in the range of 95.07%-98.16% and RSDs were between 1.23%-1.97%. CONCLUSION: It is suitable for the quality control and further studies of the herb in vivo of daodi Psoraleae Fructus-Myristicae Semen Chinese drug pair.

[Protective effects of Qizhen Jiangtang granules in diabetic nephropathy rats].

OBJECTIVE: To study the curative and protective effects of Qizhen Jiangtang Granules in the diabetic nephropathy (DN) model rats. METHODS: Healthy SD rats were fed a high-sucrose and high-fat diet and intraperitoneal injection of streptozotocin (STZ, 30 mg/kg) to establish the DN model. The rats were divided into six groups including normal control group,model group, positive control group, high-dosage group(200 mg/kg), medium-dosage group (100 mg/kg), and low-dosage group(50 mg/kg). After oral administration of Qizhen Jiangtang Granules for eight weeks, FBG,TG,TC, LDL-c, HDL-c, SCr and BUN levels in rats serum were determined, while the pathological damage of kidney tissue with PAS and HE staining were observed under microscope. RESULTS: After treatment, TG, TC, LDL-c,SCr and BUN levels were significantly decreased(P <0. 05), and HDL-c level was significantly increased(P <0. 05). The treatment also alleviated the pathological damage of kidney tissue. CONCLUSION: Qizhen Jiangtang Granules have a protective effect against kidney damage in DN model rats. The mechanism may be related to the regulation of lipid and sugar levels in serum.

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.

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.

Alteration of N6-methyladenosine epitranscriptome profile in lipopolysaccharide-induced mouse mesangial cells.

N6-Methyladenosine (m6A) is the most prevalent internal modification of messenger RNA (mRNA) in eukaryotes. The underlying molecular mechanisms of m6A modification in chronic glomerulonephritis (CGN) remain unexplored. Here, we performed methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) analyses to assess the alterations of epitranscriptome-wide m6A profile in lipopolysaccharide (LPS)-induced mouse mesangial cells (MMC). The results of our data showed 2153 significantly differential m6A peaks and 358 significantly differentially expressed genes. Furthermore, integrated analysis from MeRIP-seq and RNA-seq identified a total of 64 genes with differential m6A modification and expressed levels, of which 5 genes displayed hypermethylation and upregulation, 42 genes displayed hypermethylation and downregulation, 11 genes displayed hypomethylation and upregulation, and 8 genes displayed hypomethylation and downregulation. Many of them (including Fosl1, Sorbs1, Ambp, Fgfr3, Nedd9, Fgg, Trim13, Fgf22, Mylk, and Muc6) are implicated in the regulation of the immune and inflammatory response. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analysis found that differential 64 genes were mainly enriched in fatty acid oxidation, apoptosis signaling pathway, complement and coagulation cascades, and PPAR signaling pathway. Together, our study provided a new perspective on the understanding of molecular features of m6A modification in CGN pathogenic pathogenesis.

Identifying effective diagnostic biomarkers and immune infiltration features in chronic kidney disease by bioinformatics and validation.

Background: Chronic kidney disease (CKD), characterized by sustained inflammation and immune dysfunction, is highly prevalent and can eventually progress to end-stage kidney disease. However, there is still a lack of effective and reliable diagnostic markers and therapeutic targets for CKD. Methods: First, we merged data from GEO microarrays (GSE104948 and GSE116626) to identify differentially expressed genes (DEGs) in CKD and healthy patient samples. Then, we conducted GO, KEGG, HPO, and WGCNA analyses to explore potential functions of DEGs and select clinically significant modules. Moreover, STRING was used to analyse protein-protein interactions. CytoHubba and MCODE algorithms in the cytoscape plug-in were performed to screen hub genes in the network. We then determined the diagnostic significance of the obtained hub genes by ROC and two validation datasets. Meanwhile, the expression level of the biomarkers was verified by IHC. Furthermore, we examined immunological cells' relationships with hub genes. Finally, GSEA was conducted to determine the biological functions that biomarkers are significantly enriched. STITCH and AutoDock Vina were used to predict and validate drug-gene interactions. Results: A total of 657 DEGs were screened and functional analysis emphasizes their important role in inflammatory responses and immunomodulation in CKD. Through WGCNA, the interaction network, ROC curves, and validation set, four hub genes (IL10RA, CD45, CTSS, and C1QA) were identified. Furthermore, IHC of CKD patients confirmed the results above. Immune infiltration analysis indicated that CKD had a significant increase in monocytes, M0 macrophages, and M1 macrophages but a decrease in regulatory T cells, activated dendritic cells, and so on. Moreover, four hub genes were statistically correlated with them. Further analysis exhibited that IL10RA, which obtained the highest expression level in hub genes, was involved in abnormalities in various immune cells and regulated a large number of immune system responses and inflammation-related pathways. In addition, the drug-gene interaction network contained four potential therapeutic drugs targeting IL10RA, and molecular docking might make this relationship viable. Conclusion: IL10RA and its related hub molecules might play a key role in the development of CKD and could be potential biomarkers in CKD.

MicroRNA-339-5p inhibits lipopolysaccharide-induced rat mesangial cells by regulating the Syk/Ras/c-Fos pathway.

Chronic glomerulonephritis (CGN) is a disease occurred in glomeruli. The mechanism of CGN is regarded to be involved in a range of inflammatory responses. MicroRNA-339-5p (miR-339-5p) has been reported to be involved in inflammatory responses in many diseases. However, the role of miR-339-5p in CGN remains unclear. The purpose of this study was to investigate the role of miR-339-5p in lipopolysaccharide (LPS)-induced nephritis injury in vitro. The real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) and western blot (WB) were used to detect the expression of miR-339-5p and Syk/Ras/c-Fos pathway. Double luciferase was performed to identify targeted binding of miR-339-5p to Syk. Cell counting kit-8 (CCK-8) and flow cytometry were used to observe cell viability and cell cycle. Enzyme-linked immunosorbent assay (ELISA) was performed to measure the concentrations of inflammatory cytokines IL-1ß, IL-10, IL-6, and TNF-α. Lipopolysaccharide (LPS) could increase HBZY-1 (rat mesangial cells) cell viability, decrease the G2 phase, and promote cell proliferation and accelerate inflammatory cytokine. However, overexpression of miR-339-5p could inhibit LPS-induced HBZY-1 cell viability, decrease the expression of Syk/Ras/c-Fos signaling pathway, downregulate the expression level of inflammatory cytokines, increase the G2 phase, and inhibit cell proliferation. miR-339-5p could inhibit the proliferation and inflammation of the rat mesangial cells through regulating Syk/Ras/c-Fos signaling pathway.

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.

LncRNAs expression in adriamycin-induced rats reveals the potential role of LncRNAs contributing to chronic glomerulonephritis pathogenesis.

BACKGROUND: Chronic glomerulonephritis (CGN) is the most common form of primary glomerular disease with unclear molecular mechanisms. Currently, limited study on long non-coding RNAs (lncRNAs) in CGN is available. Our study aimed to identify potential lncRNAs and genes in the normal and adriamycin-induced CGN rats, which to explore the potential molecular mechanisms of CGN pathogenesis. METHODS: To identify LncRNAs specifically expressed in CGN, the expression of LncRNAs in glomerular tissues of rats from the adriamycin-induced group (n = 3) was compared with that in the control group (n = 3) using RNA-sequencing and real-time polymerase chain reaction (RT-PCR). Identification of differentially expressed lncRNAs and mRNAs were performed between the 2 groups. Gene ontology (GO) and pathway enrichment analyses were performed to analyze the biological functions and pathways for the differentially expressed mRNAs. LncRNA-mRNA co-expression network was constructed to analyses for the genes. The protein-protein interaction (PPI) network was visualized. RESULTS: A total of 114 significantly up-regulated and 86 down-regulated lncRNAs, 1038 up-regulated and 88 down-regulated mRNAs were identified. Additionally, Il6, with the highest connectivity degree in PPI network, was noteworthy enriched in various kinds pathways. Coding-non-coding gene co-expression networks (CNC network) were drawn based on the correlation analysis between the differentially expressed LncRNAs and mRNAs. Ten LncRNAs, NONRATT000964.2, NONRATT018086.2, NONRATT023684.2, NONRATT009530.2, NONRATT006315.2, NONRATT026805.2, MSTRG.9260.1, NONRATT009155.2, MSTRG.7681.1, NONRATT009275.2, were selected to analyze the relationship between LncRNAs and CGN via the CNC network and GO analysis. Real-time PCR result confirmed that the six LncRNAs were specifically expressed in the CGN rats. CONCLUSIONS: The ten LncRNAs were differentially expressed and might play important roles in the development of CGN. Key genes, such as Il6, Ptprc, TOP2a, Fos, Myc, etc., may be crucial biomarkers for CGN.

Effects of Qi Teng Xiao Zhuo granules on circRNA expression profiles in rats with chronic glomerulonephritis.

Objectives: To screen and study circular RNA (circRNA) expression profiles in QTXZG-mediated treatment of chronic glomerulonephritis (CGN) induced by adriamycin in rats and to research the possible roles and molecular mechanisms of QTXZG. Materials and methods: Next-generation RNA sequencing was used to identify circRNA expression profiles in CGN after QTXZG treatment compared with a CGN model group and a control group. Bioinformatics analysis was performed to predict potential target miRNAs and mRNAs. GO and pathway analyses for potential target mRNAs were used to explore the potential roles of differentially expressed (DE) circRNAs. Results: We identified 31 and 21 significantly DE circRNAs between the model group vs the control group and the model group vs the QTXZG group, respectively. Four circRNAs that resulted from the establishment of the CGN model were reversed following treatment with QTXZG. Further analysis revealed that these four circRNAs may play important roles in the development of CGN. Conclusions: This study elucidated the comprehensive expression profile of circRNAs in CGN rats after QTXZG treatment for the first time. Analysis of the circRNA-miRNA-mRNA-ceRNA network to determine potential function provided a comprehensive understanding of circRNAs that may be involved in the development of CGN. The current study indicated that therapeutic effects of QTXZG on CGN may be due to regulation of circRNA expression.