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.

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.

[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.

[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.

Urinary metabolite profiling provides potential differentiation to explore the mechanisms of adjuvant-induced arthritis in rats.

To explore the pathogenesis of rheumatoid arthritis (RA) from the perspective of metabolomics, gas chromatography time-of-flight mass spectrometry (GC-TOF/MS) technology was used to observe changes in the metabolic profiles of urine output from rats with adjuvant-induced arthritis (AA). Sprague-Dawley rats were randomly divided into a control group and an experimental group, with eight in each. Rats in the experimental group were induced by intracutaneous innoculation of 0.1 mL Freund's complete adjuvant to right paws. On day 20 after immunization, the metabolic profiles between rat control and experimental groups were compared by combining GC-TOF/MS technology with multivariate statistical approaches, including principal component analysis, partial least squares discriminant analysis and orthogonal projections to latent structures-discriminant analysis. Nine potential biomarkers were identified, including 2,2-dimethylsuccinic acid, tartronic acid, dehydroshikimic acid, hippuric acid, adenine, phenaceturic acid, l-dopa, 1,4-dihydroxy-2-naphthoic acid and melibiose. The findings indicate that the rats with AA are disturbed in metabolism of purine, amino acid, fat and energy. This study also demonstrates that the dysfunction in a range of biosynthetic and catabolic pathways, which leads to increased oxygen free radicals and inflammation, could cause underlying pathogenesis of RA. Copyright © 2016 John Wiley & Sons, Ltd.

Panax notoginseng saponins ameliorates experimental hepatic fibrosis and hepatic stellate cell proliferation by inhibiting the Jak2/ Stat3 pathways.

OBJECTIVE: To investigate the inhibitory effect of Panax notoginseng saponins (PNS) on liver fibrosis and explore the underlying mechanisms. METHODS: Carbon tetrachloride (CCl4)-treated rats and hepatic stellate cells (HSCs) were used. The effect of PNS on CCl4-induced liver fibrosis was studied with histochemical and biochemical analysis. Transforming growth factor (TGF)-ß1, α-smooth muscle actin (α-SMA), and collagen I mRNA expression were determined by reverse transcripwhile, the protein expression levels of α-SMA, collagen I, phosphorylation-Janus activated kinase signal transducer (p-Jak2)/Jak2, and phosphorylation-activator of transcription (p-Stat)3/Stat3 were determined by immunohistochemistry and/or immunoblotting. RESULTS: PNS treatment significantly improved the liver function of rats as indicated by decreased serum enzymatic activities of alanine aminotransferase and aspartate aminotransferase. Histopathological results indicated that PNS alleviated liver damage and reduced the formation of fibrous septa. Moreover, PNS significantly decreased liver hydroxyproline and significantly attenuated expressions of collagen I, α-SMA, TGF-ß1, p-Jak2 / Jak2, and p-Stat3/Stat3 in the rat liver fibrosis model and HSCs. CONCLUSION: PNS can relieve liver fibrosis by modulating Jak2/Stat3 signaling transduction pathway, which may be one of its mechanisms to suppress hepatic fibrosis.

[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.

[Changes of CD4 + CD25 + Foxp3 + Regulatory T Cells in Different Stages of COPD and Intervention of Qibai Pingfei Capsules].

OBJECTIVE: To observe the changes in proportion of CD4 + CD25 + Foxp3 + regulatory T cells (Treg) in peripheral blood of different stages of CPOD and intervention of Qibai Pingfei Capsules. METHODS: The rats were randomly divided into three groups,including normal group, COPD group and Qibai Pingfei Capsules(2. 88 g/kg)group. At the end of 7,14,21 and 28 days,eight rats were sacrificed in each group. CD4 + CD25 + Foxp3 + Treg cells in peripheral blood were measured by flow cytometry method. RESULTS: Compared with normal group, the percentages of CD4 + % in peripheral blood were not significantly different at the end of 7, 14, 21 and 28 days. However, CD4 + CD25 + % and CD4 + CD25 + Foxp3 +/CD4 + were significantly increased and CD4 + CD25 + Foxp3 + Treg% were significantly decreased at the end of 14,21 and 28 days. Compared with model group, CD4 + CD25 + %, CD4 + CD25 + Foxp3 +/CD4 + were significantly decreased, CD4 + CD25 + Foxp3 + Treg % were significantly increased at different stages of CODP. CONCLUSION: Immune disorders may exist in COPD, and Treg cells may be involved in the process of COPD. Meanwhile,the protective effect in COPD rats of Qibai Pingfei Capsules may be associated with improving the percentage of suppressive CD4 + CD25 + Foxp3 + Tregs.

[Dynamic study on curative effect of Shuganjianpifang against hepatic fibrosis induced by CCl4 in rats].

OBJECTIVE: To observe the dynamic effects of Shuganjianpifang (SGJPF) in preventing rat hepatic fibrosis induced by CCl4. METHODS: The model rat was induced by subcutaneous injection of 50% CCl4 solution in olive oil (0.1 mL/100 g), twice a week for 12 weeks. The treatment groups were given by lavage with SGJPF and Colchicine respectively once a day for 16 weeks. After 4, 8, 12 and 16 weeks, six rats were randomly sacrificed in each group. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and glutathione (GSH) in serum as well as superoxide dismutase (SOD) and malondialdehyde (MDA) in hapatic tissues were examined by colorimetric methods dynamically. Meanwhile, the histopathologic changes of hepatic tissues were observed by the HE stained method. RESULTS: Compared with normal group, the levels of ALT and AST in serum and MDA in hepatic tissues were significantly elevated, the levels of SOD in hepatic tissues and GSH in serum were significantly decreased in model group after 4, 8, 12 and 16 weeks. Compared with model group, the levels of ALT and AST in serum and MDA in hepatic tissues were significantly reduced, the levels of SOD in hepatic tissues and GSH in serum were increased significantly after 8, 12 and 16 weeks in SGJPF group and Colchicine group. Histopathologic examination results also showed that SGJPF reduced the degree of liver fibrosis after 4, 8, 12 and 16 weeks. CONCLUSION: Shuganjianpifang can obviously inhibit the formation of liver fibrosis at different phases in rats.

[Protective effects and mechanism of congguiyishen capsules on diabetic nephropathy rats].

OBJECTIVE: To study the curative and protective effects of Congguiyishen Capsules on the diabetic nephropathy (DN) model rats. METHODS: Established the DN model rats by intraperitoneal injection of urea bacteria element (Streptozotocin, STZ). The rats were divided into six groups including normal control group, model group, positive control group, high-dosage group, medium-dosage group and low-dosage group. After oral administration for 4 weeks, determined the 24 h urinary protein, Cr, kidney mass/body mass, FBG, Ang II, AT1R, AGTRAP and CTGF in the kidney. Observed the pathological damage of kidney tissue with Masson staining. RESULTS: After treatment, Cr, kidney mass index, 24 h urine protein, FBG and Ang II were decreased signicantly (P < 0.05). And the treatment also alleviated the pathological damage of kidney tissue. CONCLUSION: Congguiyishen Capsules have protective effect for DN model rats. The mechanism may be related to the suppression of inflammatory response and down-regulating the expression of AT1R, AGTRAP and CTGF.

[Optimization of processing technology for xanthii fructus by UPLC fingerprint technique and contents of toxicity ingredient].

The experiment's aim was to optimize the processing technology of Xanthii Fructus which through comparing the difference of UPLC fingerprint and contents of toxicity ingredient in water extract of 16 batches of processed sample. The determination condition of UPLC chromatographic and contents of toxicity ingredient were as follows. UPLC chromatographic: ACQUITY BEH C18 column (2.1 mm x 100 mm, 1.7 microm) eluted with the mobile phases of acetonitrile and 0.1% phosphoric acidwater in gradient mode, the flow rate was 0.25 mL x min(-1) and the detection wavelength was set at 327 nm. Contents of toxicity ingredient: Agilent TC-C18 column (4.6 mm x 250 mm, 5 microm), mobile phase was methanol-0.01 mol x L(-1) sodium dihydrogen phosphate (35: 65), flow rate was 1.0 mL x min(-1), and detection wavelength was 203 nm. The chromatographic fingerprints 16 batches of samples were analyzed in using the similarity evaluation system of chromatographic, fingerprint of traditional Chinese medicine, SPSS16.0 and SIMCA13.0 software, respectively. The similarity degrees of the 16 batches samples were more than 0.97, all the samples were classified into four categories, and the PCA showed that the peak area of chlorogenic acid, 3,5-dicaffeoylquinic acid and caffeic acid were significantly effect index in fingerprint of processed Xanthii Fructus sample. The outcome of determination showed that the toxicity ingredient contents of all samples reduced significantly after processing. This method can be used in optimizing the processing technology of Xanthii Fructus.

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.

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.

[Effect of shuganjianpifang on the expression of BCL-2 and BAX in rats livers with hepatic fibrosis].

OBJECTIVE: To observe the effect of shuganjianpifang on BCL-2, BAX expressions in livers of hepatic fibrosis rats and its mechanism. METHODS: Sixty SD rats were randomly divided into six groups. Rat liver fibrosis was induced by CCl4 twice a week for 12 weeks. Shuganjianpifang was used daily via lavage at 7th week for 6 weeks. The contents of ALT, AST, T-BiL and Alb in serum were measured. Liver samples were taken to examine the degree of liver fibrosis by HE staining. The experessions of BCL-2 and BAX were detected by immunohisto chemistry. The expression of BCL-2, BAX mRNA was detected by RT-PCR technology. RESULTS: As compared with the fibrotic model group, shuganjianpifang significantly reduced histopathological change, such as steatosis, deposition, decreased the contents of ALT, AST and T-BiL, up-regulated the expression of Alb. Meanwhile shuganjianpifang could effectively inhibit the expression of BAX, significantly enhanced the expression BCL-2 in liver fibrosis rats. CONCLUSION: Shuganjianpifang can resist hepatic fibrosis possibly by up-regulating BCL-2 expression and down-regulating BAX expression.

[Effects of extracts from ziziphi spinosae semen and schisandrae chinensis fructus on amino acid neurotransmitter in rats with insomnia induced by PCPA].

OBJECTIVE: To observe the effects of extract from Ziziphus Spinosa Semen and Schisandrae Chinensis Fructus on the content of amino acid neurotransmitter in the hypothalamus of insomnia rats induced by P-Chlorophenylalanine (PCPA) and its mechanism. METHODS: The model of insomnia rats were established by PCPA intraperitoneal injection, after the modeling, all the therapeutic group were treated with corresponding drug for one week. The hypothalamus pathological changes of the rats were observed. The contents of GABA, Glu in the hypothalamus were detected by Elisa. The GABA, Glu protein expression were detected by immunohistochemical. GABA(A), R(alpha1) and GABA(A)R(gamma2) mRNA expressions were detected by RT-PCR. RESULTS: Compared with model group, the content of GABA in the hypothalamus of rats increased obviously in the alcohol-water group (P < 0.05 or P < 0.01), while the content of Glu decreased obviously (P < 0.05 or P < 0.01). CONCLUSION: The extract from Ziziphus Spinosae Semen and Schisandrae Chinensis Fructus has obviously Sedative-hypnotic effect. Its mechanism may be related to regulating the content of amino acid neurotransmitter in the hypothalamus of rats.

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.

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.

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.