In silico prediction and validation of potential therapeutic genes in pancreatic ß-cells associated with type 2 diabetes.

Diabetes mellitus is becoming a major health burden worldwide. Pancreatic ß-cell death is a characteristic of type 2 diabetes (T2D), but the underlying mechanisms of pancreatic ß-cell death remain unknown. Therefore, the aim of the present study was to identify potential targets in the pancreatic islet of T2D. The GSE20966 dataset was obtained from the Gene Expression Omnibus (GEO) database, and differentially expressed genes (DEGs) were identified by using the GEO2R tool. The Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes Pathway enrichment analysis of DEGs were further assessed using the Database for Annotation, Visualization and Integrated Discovery. Furthermore, protein-protein interaction (PPI) networks were constructed for the up- and downregulated genes using STRING databases and were then visualized with Cytoscape. The body weight, fasting blood glucose (FBG), pancreatic index and biochemistry parameters were measured in db/db mice. Moreover, the morphology of the pancreas was detected by hematoxylin and eosin staining, and hub genes were assessed using reverse transcription-quantitative PCR (RT-qPCR) and western blot analysis. In total, 570 DEGs were screened, including 376 upregulated and 194 downregulated genes, which were associated with 'complement activation, classical pathway', 'proteolysis', 'complement activation' and 'pancreatic secretion pathway'. It was found that the body weight, FBG, alanine aminotransferase, aspartate aminotransferase, total cholesterol, triglycerides, blood urea nitrogen, creatinine, fasting serum insulin, glucagon and low-density lipoprotein cholesterol levels were significantly higher in db/db mice, while high-density lipoprotein cholesterol levels and the pancreatic index were significantly decreased. Furthermore, albumin, interleukin-8, CD44, C-C motif chemokine ligand 2, hepatocyte growth factor, cystic fibrosis transmembrane conductance regulator, histone cluster 1 H2B family member n, mitogen-activated protein kinase 11 and neurotrophic receptor tyrosine kinase 2 were identified as hub genes in PPI network. RT-qPCR and western blotting results demonstrated the same expression trend in hub genes as found by the bioinformatics analysis. Therefore, the present study identified a series of hub genes involved in the progression of pancreatic ß-cell, which may help to develop effective therapeutic strategy for T2D.

A novel identified circular RNA, circ_0000491, aggravates the extracellular matrix of diabetic nephropathy glomerular mesangial cells through suppressing miR­101b by targeting TGFßRI.

Circular RNAs (circRNAs) have crucial roles in various diseases; however, the mechanisms of action underlying circRNAs in the occurrence and development of diabetic nephropathy (DN) remains largely unknown. The present study investigated the differentially expressed circRNAs in the DN mice kidney cortex using circRNA sequencing and elucidated the role of circRNAs in mesangial cells. It was revealed that 40 circRNAs were unconventionally expressed, including 18 upregulated circRNAs and 22 downregulated circRNAs. Furthermore, circ_0000491 levels were significantly augmented in both DN mice and high glucose (HG, 30 mM)­induced mouse mesangial cells (MES13 cells). Knockdown of circ_0000491 significantly suppressed the increase of vimentin, fibronectin and α­smooth muscle actin, as well as collagen type I, III and IV, whilst reversing the decrease of E­cadherin in HG­induced MES13 cells. It was further revealed that circRNA_0000491 sponged miR­101b and that miR­101b directly targets TGFßRI. In addition, the expression levels of miR­101b were negatively associated with the transcriptional level of circRNA_0000491 and miR­101b inhibitors reversed the suppression of extracellular matrix (ECM)­associated protein synthesis mediated by knocking­down circRNA_0000491. In conclusion, the present study investigated the circRNA_0000491/miR­101b/TGFßRI axis in ECM accumulation and fibrosis­associated protein expression levels of mesangial cells, which suggested that circRNA_0000491 may be beneficial for the development of an effective therapeutic target for DN.

Ganoderma Lucidum Polysaccharides Ameliorates Hepatic Steatosis and Oxidative Stress in db/db Mice via Targeting Nuclear Factor E2 (Erythroid-Derived 2)-Related Factor-2/Heme Oxygenase-1 (HO-1) Pathway.

BACKGROUND Type 2 diabetes mellitus (T2DM) and its comorbidities, including obesity, hypertension, and hyperlipidemia, are commonly associated with non-alcoholic fatty liver disease (NAFLD). Ganoderma lucidum polysaccharide (GDLP) is one of the central bioactive components in Ganoderma lucidum with anti-inflammatory, antioxidant, and hepatoprotective properties. However, the effect and mechanisms of GDLP in hepatic steatosis remain largely unknown. In the present study, we aimed to investigate the function of GDLP in hepatic steatosis and the underlying mechanism. MATERIAL AND METHODS In this study, male db/db mice were received with a high-fat diet (HFD) to investigate the effect of GDLP in T2DM-induced hepatic steatosis. The biological characteristics of the hepatic steatosis were evaluated through the detection of clinical indicators, including biochemical parameters, histopathology, and related cytokine levels. Additionally, the protein expression levels of Nrf2 (nuclear factor E2 (erythroid-derived 2)-related factor-2) signaling pathway were investigated by using western blotting and immunohistochemical staining. RESULTS The levels of food/water intake, body weight, fasting blood glucose, plasma lipids, urinary biomarkers, hepatic lipid accumulation, and tumor necrosis factor (TNF)-alpha were observably decreased in GDLP-treated db/db mice. Additionally, administration of GDLP increased the expression of various antioxidases, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), whereas it reduced the level of malonaldehyde (MDA). Furthermore, GDLP was significantly promoted protein expression level of Nrf2 and its downstream target gene HO-1 (heme oxygenase-1) while decreased TNF-alpha expression. CONCLUSIONS These results indicate that GDLP against T2DM-induced hepatic steatosis, oxidative stress, and inflammation by improving the Nrf2/HO-1 signaling pathway in db/db mice, suggesting the GDLP may serve as an effective strategy for in fatty liver treatment.

A bioinformatics and network pharmacology approach to the mechanisms of action of Shenxiao decoction for the treatment of diabetic nephropathy.

BACKGROUND: The epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells is the main pathological alteration in diabetic nephropathy (DN). Traditional Chinese medicine (TCM) has been used for the treatment of DN in clinical practice and has been proven to be effective. PURPOSE: This aim of this study was to shed light on the efficacy of Shenxiao decoction (SXD) on the EMT of renal tubular epithelial cells and the molecular mechanisms of SXD in mice with DN, as well as on the high glucose (HG)- and TGF-ß1-induced EMT of NRK-52E and HK-2 cells. STUDY DESIGN AND METHODS: A bioinformatics and network pharmacology method were utilized to construct the active ingredient-target networks of SXD that were responsible for the beneficial effects against DN. The effects of RUNX3 were validated in HG- and TGF-ß1-induced EMT processes in NRK-52E and HK-2 cells. RESULTS: Bioinformatics analysis revealed that 122 matching targets were closely associated with the regulation of cell migration and the AGE-RAGE signaling pathway in diabetic complications. The results also revealed that, relative to the mice with DN, the mice in the treatment group had an improved general state and reduced blood glucose levels. The degradation of renal function was ameliorated by SXD. Moreover, the protective effects of SXD were also observed on renal structural changes. Furthermore, SXD suppressed the activation of the transforming growth factor (TGF)-ß1/Smad pathway and upregulated the RUNX3 and E-cadherin levels and downregulated the extracellular matrix (ECM) protein levels in mice with DN. SXD was further found to prevent the HG- and TGF-ß1-induced EMT processes in NRK-52E and HK-2 cells. Additionally, the overexpression of RUNX3 markedly inhibited the EMT and TGF-ß1/Smad pathway induced by HG and TGF-ß1 in NRK-52E and HK-2 cells. CONCLUSION: Taken together, these results suggest that SXD maybe alleviate EMT in DN via the inhibition of the TGF-ß1/Smad/RUNX3 signaling pathway under hyperglycemic conditions.

Effect of Grilled Nux Vomica on Differential RNA Expression Profile of Gastrocnemius Muscle and Toll­Like Receptor 4 (TLR-4)/Nuclear Factor kappa B (NF-κB) Signaling in Experimental Autoimmune Myasthenia Gravis Rats.

BACKGROUND Myasthenia gravis (MG) is a progressive autoimmune disorder caused by the production of antibodies directed against acetylcholine receptors (AChRs), resulting in muscle weakness and fatigue. This study aimed to explore the effect and mechanism of grilled nux vomica (GNV) in experimental autoimmune myasthenia gravis (EAMG) rats. MATERIAL AND METHODS Rat 97-116 peptides were used to mediate disease in the EAMG model in SPF female Lewis rats. The treatment groups received grilled nux vomica (75 mg/kg, 150 mg/kg, and 225 mg/kg). The autoantibody and inflammatory cytokines levels were measured by enzyme-linked immunosorbent assay (ELISA). RNA profiling was performed on high-dose and model group rats. Profiling results and TLR-4/NF-kappaB signaling were validated by q-PCR and Western blot analysis. RESULTS The results showed that GNV could attenuate the symptoms of EAMG rats. There was a decreased level of AChR-ab, IFN-γ, TNF-alpha, IL-2, IL-4, and IL-17 levels, and an increased level of TGF-ß1. In total, 235 differentially expressed genes (DEGs), consisting of 175 upregulated DEGs and 60 downregulated DEGs, were identified. Functional annotation demonstrated that DEGs were largely associated with leukocyte cell-cell adhesion, NF-kappa B signaling pathway, muscle contraction, and cardiac muscle contraction pathway. Rac2, Itgb2, Lcp2, Myl3, and Tnni1 were considered as hub genes with a higher degree value in the protein-protein interaction (PPI) network. The q-PCR and Western blot results of hub genes were consistent with RNA profiles. GNV treatment also significantly reduced the TLR-4 and NF-kappaB p65 protein expression in EAMG rats. CONCLUSIONS These results indicate that grilled nux vomica ameliorates EAMG by depressing the TLR-4/NF-kappaB signaling pathway, and hub genes may serve as potential targets for MG treatment.

Identification of potential therapeutic target genes in mouse mesangial cells associated with diabetic nephropathy using bioinformatics analysis.

The aim of the present study was to identify genes under the effect of transforming growth factor-ß (TGF-ß1), high glucose (HG) and glucosamine (GlcN) in MES-13 mesangial cells and elucidate the molecular mechanisms of diabetic nephropathy (DN). The gene expression datasets GSE2557 and GSE2558 were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were independently screened using the GEO2R online tool. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the Database for Annotation, Visualization, and Integrated Discovery. The protein-protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes and Cytoscape software. The hub genes were identified by the NetworkAnalyzer plugin. Overlapping genes were subjected to molecular docking analysis using SystemsDock. A total of 202 upregulated and 158 downregulated DEGs from the HG-treated groups, 138 upregulated and 103 downregulated DEGs from the GlcN-treated groups, and 81 upregulated and 44 downregulated DEGs from the TGF-ß1-treated groups were identified. The majority of the DEGs were independently enriched in 'nucleosome assembly', 'chromatin silencing' and 'xenobiotic glucuronidation'. In addition, KEGG pathways were significantly enriched in 'systemic lupus erythematosus', 'protein processing in endoplasmic reticulum' and 'aldarate metabolism pathway', and 'TNF signaling pathway' intersected in the TGF-ß1-treated and HG-treated groups. In total, eight hub genes, Jun, prostaglandin-endoperoxide synthase 2 (Ptgs2), fibronectin 1 (Fn1), cyclin-dependent kinase (Cdk)2, Fos, heat shock protein family A (Hsp70) member 5 (Hspa5), Hsp90b1 and homo sapiens hypoxia upregulated 1 (Hyou1), and three overlapping genes, Ras homolog gene family, member B (RHOB), complement factor H (CFH) and Krüppel-like factor 15 (KLF15), were selected. Valsartan with RHOB, and fosinopril with CFH and KLF15 had preferential binding activity. In conclusion, Jun, Ptgs2, Fn1, Cdk2, Fos, Hspa5, Hsp90b1, Hyou1, RHOB, CFH and KLF15 may be potential therapeutic targets for mesangial cells associated with DN, which may provide insight into DN treatment strategies.

[Mechanism of effective components of Mori Folium in alleviating insulin resistance based on JNK signaling pathway].

A stable hepatoma cell line(Hep G2 cell) insulin resistance model was established and used to analyze the effect of effective components of Mori Folium in alleviating insulin resistance,and preliminary explore the mechanism for alleviating insulin resistance. The Hep G2 insulin action concentration and the duration of action were investigated using the glucose oxidase method(GOD-POD method) to establish a stable Hep G2 insulin resistance model. Normal control group,model group,Mori Folium polysaccharide group,Mori Folium flavonoid group and rosiglitazone group were divided to determine the glucose consumption. The effect of Mori Folium effective components on Hep G2 insulin resistance was analyzed. The mRNA expressions of JNK,IRS-1 and PDX-1 in each group were detected by Real-time quantitative PCR(qRT-PCR). The protein expressions of p-JNK,IRS-1 and PDX-1 were detected by Western blot. And the mechanism of effective components of Mori Folium in alleviating insulin resistance was investigated. The results showed that the glucose consumption was significantly decreased in the insulin resistance cells after incubation with 25. 0 mg·L-1 insulin for 36 h(P<0. 01),and the model was relatively stable within 36 h. Mori Folium polysaccharides and flavonoids all alleviated insulin resistance,among which Mori Folium flavonoids had better effect in alleviating Hep G2 insulin resistance(P<0. 05). The qRT-PCR analysis showed that Mori Folium polysaccharides and flavonoids could inhibit JNK and IRS-1 mRNA expressions,while enhancing PDX-1 mRNA expression. Western blot analysis displayed that Mori Folium polysaccharides and flavonoids could inhibit p-JNK and IRS-1 protein expressions,while enhancing PDX-1 protein expression. Mori Folium polysaccharides and flavonoids can alleviate insulin resistance in Hep G2 cells,and its mechanism may be the alleviation of insulin resistance by inhibiting JNK signaling pathway.

Extraction of flavonoids from Chrysanthemum morifolium and antitumor activity in vitro.

The aim of the present study was to optimize flavonoid extraction from Chrysanthemum morifolium and to study the antitumor effects of flavonoids on human gastric cancer MKN45 cells in vitro. A single factor experiment was designed and the extraction process was optimized using an orthogonal test. MKN45 cells were treated with different concentrations of flavonoid from Chrysanthemum morifolium for 24 and 48 h and the inhibitory effect on the MKN45 cells was evaluated using an MTT assay. Following staining with Annexin V-fluorescein isothiocyanate/propidium iodide, flow cytometry was performed. The optimized flavonoid extraction conditions were as follows: Duration of ultrasonic treatment: 35 min; ethanol concentration: 75%; extraction temperature: 80°Cand liquid-to-solid ratio 25: 1. Under the above conditions, the extraction rate of flavonoids was 5.24%. When compared with a blank control group, flavonoids extracted from Chrysanthemum morifolium inhibited the proliferation of MKN45 cells in a dose- and time-dependent manner. Furthermore, in cell groups treated with low, moderate and high concentrations of flavonoid, it was observed that the proportion of apoptotic cells increased in a dose-dependent manner. The extraction process optimized by the orthogonal test achieved a high yield and satisfactory extraction efficiency. Additionally, the experiment demonstrated that flavonoids from Chrysanthemum morifolium inhibited the growth of MKN45 cells and induced their apoptosis. Thus, flavonoids from Chrysanthemum morifolium exerted antitumor effects on MKN45 cells, which may be exploited as a potential antitumor therapeutic for gastric cancer.

Serum TGF-ß1 as a Biomarker for Type 2 Diabetic Nephropathy: A Meta-Analysis of Randomized Controlled Trials.

BACKGROUND: Abnormal expression of serum TGF-ß1 was found in patients with diabetic nephropathy. However, the association of TGF-ß1 with the risk of diabetic nephropathy remains unknown. The present study was undertaken to investigate whether such an association exists. METHODS: We searched the Chinese VIP, Wangfang, China National Knowledge Infrastructure, PubMed, Embase, and Google Scholar databases for relevant studies and extracted all eligible data. Stata12 software was used for statistical analysis. RESULTS: Nine reports met our criteria and were used for data extraction. There were 264 patients and 227 healthy controls from qualified reports in this meta-analysis. The results suggested that serum TGF-ß1 levels were significantly up-regulated in patients with diabetic nephropathy; the instrumental variable was 3.94 (95% confidence interval 3.20-4.68, p<0.01). CONCLUSIONS: Meta-analysis suggested that elevated serum TGF-ß level in patients with diabetes is associated with a high risk of nephropathy. Further studies are required to validate these observations.

Study on the relationship between Chinese medicine constitutive susceptibility and diversity of syndrome in diabetic nephropathy.

OBJECTIVE: To explore the relationship between Chinese medicine (CM) constitutive susceptibility and syndrome diversity in diabetic nephropathy (DN). METHODS: Epidemiologic investigation on constitution adopting the "Constitution in Chinese Medicine Questionnaire" (CCMQ), and survey on syndrome type by CM syndrome scale (preliminary) were carried out in 180 DN patients. Cluster analysis on symptom items was used to determine the syndrome type, and canonical correlation analysis was used to analyze the relationship between patients' constitution and syndrome. RESULTS: Baseline levels in all enrolled patients were not different statistically. Cluster analysis showed 8 syndromes existed in DN patients, namely: I, qi-yin deficiency with qi-stagnancy type; II, yin-yang deficiency with heat-water-blood stasis type; III, qi-yin deficiency with dampness-heat type; IV, yin-yang deficiency with blood-stasis and heat type; V, qi-yin deficiency with stagnant heat type; VI, yin-yang deficiency with inner dampness-heat stagnancy type; VII, yin deficiency with heat stagnancy type; and VIII, Kidney (Shen)-Spleen (Pi) deficiency with stagnant heat type. Correlation analysis on the 8 syndromes and the 9 constitutions showed statistical significant correlations between syndrome III and dampness-heat constitution (P=0.0001); syndrome IV and blood-stasis constitution (P=0.0001); and syndrome VII and yin-deficiency constitution (P=0.0180). CONCLUSION: Certain relationship revealed between CM constitutions and syndrome types; constitution decides the disease genesis, its syndrome type and prognosis, as well as the change of syndromes.