Combined Plasma Olink Proteomics and Transcriptomics Identifies CXCL1 and TNFRSF12A as Potential Predictive and Diagnostic Inflammatory Markers for Acute Kidney Injury.

Acute kidney injury (AKI) poses a significant global public health challenge. Current methods for detecting AKI rely on monitoring changes in serum creatinine (Scr), blood urea nitrogen (BUN), urinary output and some commonly employed biomarkers. However, these indicators are usually neither specific nor sensitive to AKI, especially in cases of mild kidney injury. AKI is accompanied by severe inflammatory reactions, resulting in the upregulation of numerous inflammation-associated proteins in the plasma. Plasma biomarkers are a noninvasive method for detecting kidney injury, and to date, plasma inflammation-associated cytokines have not been adequately studied in AKI patients. The objective of our research was to identify novel inflammatory biomarkers for AKI. We utilized Olink proteomics to analyze the alterations in plasma inflammation-related proteins in the serum of healthy mice (n = 2) or mice treated with cisplatin (n = 6). Additionally, transcriptome datasets for the lipopolysaccharide (LPS), cisplatin, and ischemia‒reperfusion injury (IRI) groups were obtained from the National Center of Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database. We calculated the intersection of differentially expressed proteins (DEPs) and genes (DEGs) from both datasets. In the Olink proteomics analysis, the AKI group had significantly greater levels of 11 DEPs than did the control group. In addition, 56 common upregulated DEGs were obtained from the transcriptome dataset. The expression of CXCL1 and TNFRSF12A overlapped across all the datasets. The transcription and protein expression levels of CXCL1 and TNFRSF12A were detected in vivo. The gene and protein levels of CXCL1 and TNFRSF12A were significantly increased in different AKI mouse models and clinical patients, suggesting that these genes and proteins could be potential specific biomarkers for the identification of AKI.

Integrative analysis of single-cell and bulk RNA-sequencing data revealed T cell marker genes based molecular sub-types and a prognostic signature in lung adenocarcinoma.

Immunotherapy has emerged as a promising modality for addressing advanced or conventionally drug-resistant malignancies. When it comes to lung adenocarcinoma (LUAD), T cells have demonstrated significant influence on both antitumor activity and the tumor microenvironment. However, their specific contributions remain largely unexplored. This investigation aimed to delineate molecular subtypes and prognostic indicators founded on T cell marker genes, thereby shedding light on the significance of T cells in LUAD prognosis and precision treatment. The cellular phenotypes were identified by scrutinizing the single-cell data obtained from the GEO repository. Subsequently, T cell marker genes derived from single-cell sequencing analyses were integrated with differentially expressed genes from the TCGA repository to pinpoint T cell-associated genes. Utilizing Cox analysis, molecular subtypes and prognostic signatures were established and subsequently verified using the GEO dataset. The ensuing molecular and immunological distinctions, along with therapy sensitivity between the two sub-cohorts, were examined via the ESTIMATE, CIBERSORT, and ssGSEA methodologies. Compartmentalization, somatic mutation, nomogram development, chemotherapy sensitivity prediction, and potential drug prediction analyses were also conducted according to the risk signature. Additionally, real-time qPCR and the HPA database corroborated the mRNA and protein expression patterns of signature genes in LUAD tissues. In summary, this research yielded an innovative T cell marker gene-based signature with remarkable potential to prognosis and anticipate immunotherapeutic outcomes in LUAD patients.

Identification of Cell-Cell Communications by Single-Cell RNA Sequencing in End Stage Renal Disease Provides New Insights into Immune Cell Heterogeneity.

Objective: Impaired immune system characterized by low-grade inflammation is closely associated with kidney chronic kidney disease (CKD) progression. To reveal the alterations of the function, component, and intercellular communication of immune cells during the progression of CKD. Patients and Methods: We conducted a case-control study enrolling regular hemodialysis patients and healthy controls. Clinical data, serum and peripheral blood mononuclear cell (PBMC) samples were collected. Flow cytometry and single-cell RNA sequencing were performed to quantitatively analyze the immune cell subsets and T-cell subsets of PBMCs. scRNA data of GSE140023 containing mouse unilateral ureteral obstruction (UUO) models were analyzed the heterogeneity of immune cells. Results: Overall reduction in peripheral blood lymphocyte subsets in patients with end-stage renal disease (ESRD) was observed. A higher ratio of Th17/Treg, Th1/Treg, and b-cell/Treg in the ESRD group was associated with a decrease in eGFR, PTH, and ferritin. Among T cell subsets identified by scRNA analysis, Th17 cells were significantly increased in the ESRD and UU0 group. TFH, Th1, and Th2 cells are located at the final stage in the developmental tree, while Treg and memory CD8+ T cells are at the beginning site. Early developmental differentiation of Th17, Th1, and Tfh cells was observed in the ESRD and UUO group. Analysis of intercellular communication between t-cell subpopulations identified two major input and output signaling pathways: the CD40 and macrophage inhibitory factor (MIF) pathways. The MIF signaling pathway primarily mediates intercellular communication among th17 effects, CD8+ t-cell, and Th17-Treg in the ESRD group, the serum level of MIF showed significant upregulation, which was closely related to Th17/Treg cells. Conclusions: A global immune imbalance was closely associated with the deterioration in renal function and complication development. The MIF signaling pathway mediates Th17/Treg communication and promotes the trans-differentiation of Treg cells to Th17 cells in CKD progression.

Effects of Liver-Regulating Herb Compounds on Varicocele-Associated Testicular Dysfunction Through Restoring Hormones and Spermatocytes Apoptosis.

PURPOSE: Varicocele is considered one of the causes of male infertility. Though varicocelectomy is supposed to improve semen parameters in adult infertile men, some patients with varicocele were still infertile after varicocelectomy. Previous studies showed Traditional Chinese Medicine, Liver-regulating herb compounds (LRHC) could improve the semen quality and increase fertility rates of infertile patients with varicocele. This study aimed to throw light on the mechanism of LRHC on varicocele-associated infertility. MATERIALS AND METHODS: Rats with varicocele-induced were treated with LRHC at dosage of 1mL/100g by intragastric administration for 90 days. The effects of LRHC on hormones and spermatocytes apoptosis were examined using ELISA assay, Western blotting, and flow cytometry. RESULTS: Rats induced with varicocele showed a higher level of follicle stimulating hormone (FSH) in serum, which was brought back to normal level by LRHC. After treatment with LRHC, both testicular tissue in vivo and Sertoli cell TM4 cells in vitro showed elevated expressions of FSHR. Cell viabilities of TM4 cells and spermatocyte GC-2 cells were improved by LRHC treatment under normoxia and hypoxia conditions. Moreover, LRHC protected GC-2 cells from apoptosis induced by hypoxia. The expression of Bax reduced, while that of Bcl-2 increased after treatment with LRHC. CONCLUSION: This study revealed that LRHC had protective effects on spermatogenic disturbance caused by varicocele through regulating hormones and reducing spermatogenic cell apoptosis under hpoxia conditions.

A Disintegrin and Metalloproteinase 10 (ADAM10) Is Essential for Oligodendrocyte Precursor Development and Myelination in the Mouse Brain.

A disintegrin and metalloproteinase 10 (ADAM10) plays an essential role in the regulation of survival, proliferation, migration, and differentiation of various neural cells. Nevertheless, the role of ADAM10 in oligodendrocyte precursors (OPCs) and myelination in the central nervous system (CNS) of developing and adult mouse brains is still unknown. We generated ADAM10 conditional knockout (ADAM10 cKO) mice lacking the ADAM10 gene primarily in OPCs by crossing NG2-Cre mice with ADAM10 loxp/loxp mice. We found that OPCs expressed ADAM10 in the mouse corpus callosum and the hippocampus. ADAM10 cKO mice showed significant loss of back hair and reduction in weight and length on postnatal (30 ± 2.1) day, died at (65 ± 5) days after birth, and exhibited the "anxiety and depression-like" performances. Conditional knockout of ADAM10 in OPCs resulted in a prominent increase in myelination and a decrease in the number of OPCs in the corpus callosum at P30 owing to premyelination and lack of proliferation of OPCs. Moreover, the number of proliferating OPCs and mature oligodendrocytes (OLs) also decreased with age in the corpus callosum of ADAM10 cKO mice from P30 to P60. Western blot and RT-PCR results showed that the activation of Notch-1 and its four target genes, Hes1, Hes5, Hey1, and Hey2, was inhibited in the corpus callosum tissue of ADAM10 knockout mice. In our study, we provided experimental evidence to demonstrate that ADAM10 is essential for modulating CNS myelination and OPC development by activating Notch-1 signaling in the developing and adult mouse brain.

Mechanism of hydroxysafflor yellow A on acute liver injury based on transcriptomics.

Objective: To investigate how Hydroxysafflor yellow A (HSYA) effects acute liver injury (ALI) and what transcriptional regulatory mechanisms it may employ. Methods: Rats were randomly divided into five groups (n = 10): Control, Model, HSYA-L, HSYA-M, and HSYA-H. In the control and model groups, rats were intraperitoneally injected with equivalent normal saline, while in the HSYA groups, they were also injected with different amounts of HSYA (10, 20, and 40 mg/kg/day) once daily for eight consecutive days. One hour following the last injection, the control group was injected into the abdominal cavity with 0.1 ml/100 g of peanut oil, and the other four groups got the same amount of a peanut oil solution containing 50% CCl4. Liver indexes were detected in rats after dissection, and hematoxylin and eosin (HE) dyeing was utilized to determine HSYA's impact on the liver of model rats. In addition, with RNA-Sequencing (RNA-Seq) technology and quantitative real-time PCR (qRT-PCR), differentially expressed genes (DEGs) were discovered and validated. Furthermore, we detected the contents of anti-superoxide anion (anti-O2 -) and hydrogen peroxide (H2O2), and verified three inflammatory genes (Icam1, Bcl2a1, and Ptgs2) in the NF-kB pathway by qRT-PCR. Results: Relative to the control and HSYA groups, in the model group, we found 1111 DEGs that were up-/down-regulated, six of these genes were verified by qRT-PCR, including Tymp, Fabp7, Serpina3c, Gpnmb, Il1r1, and Creld2, indicated that these genes were obviously involved in the regulation of HSYA in ALI model. Membrane rafts, membrane microdomains, inflammatory response, regulation of cytokine production, monooxygenase activity, and iron ion binding were significantly enriched in GO analysis. KEGG analysis revealed that DEGs were primarily enriched for PPAR, retinol metabolism, NF-kB signaling pathways, etc. Last but not least, compared with the control group, the anti-O2 - content was substantially decreased, the H2O2 content and inflammatory genes (Icam1, Bcl2a1, and Ptgs2) levels were considerably elevated in the model group. Compared with the model group, the anti-O2 - content was substantially increased, the H2O2 content and inflammatory genes (Icam1, Bcl2a1, and Ptgs2) levels were substantially decreased in the HSYA group (p < 0.05). Conclusion: HSYA could improve liver function, inhibit oxidative stress and inflammation, and improve the degree of liver tissue damage. The RNA-Seq results further verified that HSYA has the typical characteristics of numerous targets and multiple pathway. Protecting the liver from damage by regulating the expression of Tymp, Fabp7, Serpina3c, Gpnmb, Il1r1, Creld2, and the PPAR, retinol metabolism, NF-kappa B signaling pathways.

[To explore mechanism of Scabiosa comosa against liver fibrosis based on pharmacodynamics and network pharmacology].

This study aims to systematically elucidate the pharmacodynamics and network pharmacological mechanism of Mongolian medicinal plants Scabiosa comosa, explore their key targets and related pathways, and further clarify the mechanism of the plants in treating liver fibrosis. Wistar rats were assigned into the blank group, carbon tetrachloride-induced liver fibrosis model group, and low-, medium-, and high-dose S. comosa groups. HE staining and Masson staining were performed for the observation of liver tissue under a microscope. Further, Wistar rats were assigned into a control group and a S. comosa group for administration. Seven days later, blood was collected from the abdominal aorta, and different doses of drug-containing serum samples were used to treat hepatic stellate cell-T6(HSC-T6). Flow cytometry was adopted to detect the apoptosis of HSC-T6 cells. Ultra-high performance liquid chromatography-time of flight-mass spectrometry(UHPLC-TOF-MS) was employed to determine the components in Scabiosa comosa. The target of S. comosa and liver fibrosis were obtained from SwissTargetPrediction and GeneCards, respectively, and the common targets were selected as the anti-liver fibrosis targets. Protein-protein interaction was analyzed via STRING. Gene Ontology(GO) annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment were carried out via Metascape. Phosphatidylinosital 3-kinase(PI3 K), protein kinase B(AKT), p-AKT, p38, and p-p38 targets which are involved in the top-ranked PI3 K/AKT and mitogen activated kinase-like protein(MAPK) signaling pathways were selected for validation via Western blot. The HE and Masson staining results showed that Scabiosa alleviated the hyperplasia of connective tissue and the fibrosis. The serum containing Scabiosa significantly promoted the apoptosis of HSC-T6 in a concentration-dependent manner. A total of 76 chemical components were identified by UHPLC-TOF-MS, among which flavonoids, alkaloids, terpenoids, phenols, and fatty acids were the main components. According to the prediction, there were 63 anti-liver fibrosis targets in Scabiosa comosa, the annotated GO terms of which involved biological processes, cell components, and molecular functions. The KEGG pathway enrichment showed that the targets were mainly involved in PI3 K/AKT, epidermal growth factor receptor(EGFR), RAS-associated protein 1(Rap1), hypoxia-inducible factor 1(HIF-1), resistance to audiogenic seizures(Ras), and MAPK signaling pathways. Western blot results showed that compared with the model group, S. comosa down-regulated the protein levels of α-smooth muscle actin(α-SMA), collagen Ⅰ, PI3 K, AKT, p-AKT, p38, and p-p38 in liver tissue. Compared with the control group, the low-, medium-, and high-dose S. comosa significantly down-regulated the protein levels of α-SMA, collagen Ⅰ, PI3 K, AKT, p-AKT, p38, and p-p38 in HSC-T6. The evidence of pharmacodynamics, network pharmacology, and molecular biology indicated that the plants of S. comosa had significant activity against liver fibrosis, the mechanism of which may involve the regulation of the key targets PI3 K, AKT, and MAPK14 p38 in the PI3 K/AKT and MAPK signaling pathways.

Cyclophosphamide Attenuates Fibrosis in Lupus Nephritis by Regulating Mesangial Cell Cycle Progression.

OBJECTIVES: Most patients with systemic lupus erythematosus (SLE) develop lupus nephritis (LN) with severe kidney manifestations. Renal fibrosis can be primarily attributed to overproliferation of mesangial cells (MCs), which are subject to drug treatment. Nevertheless, the detailed mechanisms remain elusive. We sought to identify the effect of cyclophosphamide (CTX), a drug commonly used for LN treatment, on MC proliferation and explore its underlying mechanisms. Material/Methods. Cell proliferation and fibrosis in mouse kidney tissues were determined by histopathology staining techniques. Flow cytometry was used for cell cycle analysis. Cell cycle regulators were examined in vitro following treatment of immortalized human MCs with platelet-derived growth factor subunit B (PDGF-B). Quantitative real-time PCR and western blot analyses were used to measure the mRNA and protein levels of candidate cell cycle regulators, respectively. RESULTS: CTX inhibited cell overproliferation induced by platelet-derived growth factor subunit B in vitro and in vivo. CTX (40 mg/l) was sufficient to induce G0/G1 phase cell cycle arrest. CTX treatment downregulated many critical cell cycle regulators including cyclins and cyclin-dependent kinases but upregulated cyclin-dependent kinase inhibitors. Additionally, CTX-treated samples showed significantly reduced fibrosis, as indicated by lower expression of interleukin-1ß and α-smooth muscle actin. CONCLUSION: CTX inhibits proliferation of MCs by modulating cell cycle regulator and therefore arresting them at G1 phase. CTX treatment significantly alleviates the severity of renal fibrosis. These findings provide novel insights into the mechanisms by which CTX affects LN.

Study on the effect of Mongolian medicine Qiwei Qinggan Powder on hepatic fibrosis through JAK2/STAT3 pathway.

This research aimed to evaluate the antihepatic fibrosis effect and explore the mechanism of Qiwei Qinggan Powder (QGS-7) in vivo and in vitro. Carbon tetrachloride (CCl4)-treated rats and hepatic stellate cells (HSCs) were used. QGS-7 treatment significantly improved the liver function of rats as indicated by decreased serum enzymatic activities of alanine aminotransferase, aspartate transaminase, and alkaline phosphatase. Meanwhile, the hydroxyproline of liver was significantly decreased. Histopathological results indicated that QGS-7 alleviated liver damage and reduced the formation of fibrosis septa. Moreover, QGS-7 significantly attenuated expressions of Alpha smooth muscle actin, Collagen I, Janus kinase 2 (JAK2), phosphorylation-JAK2, signal transducer and activator of transcription 3 (STAT3), phosphorylation-STAT3 in the rat hepatic fibrosis model. QGS-7 inhibited HSC proliferation and promoted it apoptosis. QGS-7 may affect hepatic fibrosis through JAK2/STAT3 signaling pathway so as to play an antihepatic fibrosis role.

Association between serum irisin concentrations and sarcopenia in patients with liver cirrhosis: a cross-sectional study.

Sarcopenia is an independent predictor of mortality in patients with liver cirrhosis. However, evidence has emerged that skeletal muscles mediate their protective effect against sarcopenia by secreting myokines. Therefore, we investigated whether irisin was associated with sarcopenia in patients with liver cirrhosis. This was an observational cross-sectional study of data collected from 187 cirrhotic patients. Sarcopenia was defined by computed tomography (CT) scans using specific cutoffs of the 3rd lumbar vertebra skeletal muscle index (L3 SMI). Morning irisin levels were obtained in all patients. Of the 187 patients, sarcopenia was noted in 73 (39%). Irisin concentrations were lower in sarcopenic patients (32.40 pg/ml [interquartile range (IQR): 18.70, 121.26], p < 0.001) than in nonsarcopenic patients. There was a weak correlation between L3 SMI and irisin levels (r = 0.516, p < 0.001). Multivariable regression analysis including L3 SMI, body mass index (BMI), very-low-density lipoprotein (VLDL)-cholesterol, aspartate aminotransferase (AST), adiponectin, and irisin levels showed that L3 SMI (odds ratio [OR] = 0.915, p = 0.023), adiponectin levels (OR = 1.074, p = 0.014), irisin levels (OR = 0.993, p < 0.001) and BMI (OR = 0.456, p = 0.004) were independently associated with sarcopenia. Irisin levels are associated with sarcopenia in patients with liver cirrhosis. This paper addresses a gap in the literature and facilitates the future transition into clinical treatment.

HnRNPA2/B1 Is a Novel Prognostic Biomarker for Breast Cancer Patients.

Aims: Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1) is highly expressed in multiple types of tumor tissues and could potentially be used as a biomarker for the early detection of lung cancer. However, there is little evidence supporting its clinical significance as a prognostic marker in breast cancer. Materials and Methods: We retrospectively analyzed the protein expression and localization of hnRNPA2/B1 protein in breast cancer tissues and adjacent normal tissues from 50 patients with Stage II and III breast cancer who were treated at Shanxi Provincial People's Hospital from May 2018 to May 2019 using western blot, and immunofluorescent and immunohistochemical staining assays. In addition, bioinformatic analyses using the Affymetrix Human Genome database were performed to examine the mRNA levels of hnRNPA2/B1 in normal and breast cancer tissues, and to determine their correlation with the survival rates of breast cancer patients. Results: Based on the cohort of 50 patients, HnRNPA2/B1 protein was expressed in both the cytoplasm and nucleus of breast cancer cells. The protein levels of hnRNPA2/B1 in breast cancer tissues were significantly higher than those in adjacent normal tissues (p < 0.001). Furthermore, bioinformatic analyses of hnRNPA2/B1 mRNA expression levels demonstrated that they were negatively correlated with overall survival and disease-specific survival rates in breast cancer patients. Conclusion: Our study indicates that hnRNPA2/B1 could serve as a novel prognostic biomarker for breast cancer.

Effects of liver-regulating herb compounds on testicular morphological and ultrastructural changes in varicocele rats through SCF/C-KIT pathway.

Liver-regulating herb compound (LRHC) has good effects on improving sperm quality and male fertility of varicocele (VC) patients. But the mechanism of LRHC on VC is still not clear. This study explored the effects of LRHC on histomorphological and ultrastructural changes and expression of stem cell factor (SCF) and C-KIT of VC rat testis. Twenty-four male rats were divided into three groups with eight rats in each group as sham, varicocele and LRHC groups. Testis specimens were collected for light microscopy and transmission electron microscopy respectively. The expression of SCF/C-KIT was detected with Western blot. Results showed that seminiferous tubules in VC rats were damaged and cell numbers were decreased. Ultrastructural alterations were observed, such as increased thickness of lamina propria, vacuolation in Sertoli cells, spermatocytes and spermatids, and abnormal head and mitochondria in spermatozoa. While in LRHC-treated rats, the architectures of seminiferous tubules were as organised and compact as that of sham animals, and ultrastructure of Sertoli, Leydig and germ cells developed well. LRHC ameliorated histological appearance and ultrastructure by VC. In addition, the abnormal expression of SCF and C-KIT were observed in testicular tissues from rats with VC, which were brought back to normal level by LRHC.

Anti-liver fibrosis effect of total flavonoids from Scabiosa comosa Fisch. ex Roem. et Schult. on liver fibrosis in rat models and its proteomics analysis.

BACKGROUND: To explore the potential therapeutic effect of total flavonoids (TFs) extracted from Scabiosa comosa Fisch. ex Roem. et Schult on liver fibrosis in rat models and to identify the possible targets and pathways of TF in treating liver fibrosis by using a quantitative proteomics method. METHODS: Sixty Wistar rats were equally randomized into five groups: a blank control group, a model group, and high-, intermediate-, and low-dose TF treatment groups. Except for the blank control group, rats in the other four groups were intragastrically administered with CCL4 2 mL/kg to establish the liver fibrosis models. Furthermore, the high-, intermediate-, and low-dose TF groups were intragastrically given TF at a dose of 200, 100 and 50 mg/kg, respectively. After 10 weeks, the rats were sacrificed, and blood and liver samples were collected. Serum alanine transaminase (ALT), Aspartate aminotransferase (AST), and alkaline phosphatase (ALP) levels were measured, and hematoxylin and eosin (HE) staining and Masson's trichrome staining were used to observe the pathological changes in each group. The hydroxyproline content was also determined. Real-time polymerase chain reaction (PCR) and Western blotting (WB) were performed to detect the mRNA and protein expressions of α-smooth muscle actin (αSMA) and Collagen I. Mass spectrometry was performed for proteomic analysis. RESULTS: Compared with the blank control group, the model group had significantly higher ALT, AST, ALP, and hydroxyproline levels; also, HE and Masson staining showed fibrotic lesions and inflammatory cell infiltration in the model group. Compared with the model group, the high-, intermediate-, and lowdose TF groups had significantly decreased ALT, AST, and ALP levels (P<0.05), and a significantly lower hydroxyproline level (P<0.05), along with remarkably improved fibrotic lesions and inflammatory cell infiltration. Real-time PCR and WB showed that the model group had significantly higher expressions of αSMA and collagen I than those in the blank control group, whereas the TF groups had significantly lower expressions of αSMA and collagen I than those in the model group. A total of 5,014 proteins were detected by quantitative proteomics, among which 205 proteins were differentially expressed, 77 of which were upregulated and 128 of which were down-regulated. KEGG pathway analysis indicated that the peroxisome proliferator activated receptor (PPAR) and ECM-receptor interaction pathways were down-regulated in the TF groups compared with the model group. Among them, fatty-acid-binding protein (FABP) and von Willebrand factor (vWF) were the key proteins in the PPAR and extracellular matrix (ECM)-receptor interaction pathways. The proteomic results were validated by using WB, yielding consistent results. CONCLUSIONS: Our result demonstrated that the TF extract of Scabiosa comosa Fisch. ex Roem. et Schult has a good anti-liver fibrosis effect and may prevent liver fibrosis by reducing the content of α-SMA, CollagenⅠ in liver tissue. The anti-fibrosis mechanism of TF extract of Scabiosa comosa Fisch. ex Roem. et Schult may be the inhibition of key proteins FABP and vWF in PPAR, ECM RECEPTOR INTERACTION pathway.

Downregulation of PTEN promotes podocyte endocytosis of lipids aggravating obesity-related glomerulopathy.

With the increasing prevalence of obesity in adults worldwide, the incidence of obesity-related glomerulopathy (ORG) has increased yearly, becoming one of the leading causes of end-stage renal disease. Studies have demonstrated significant correlations between hyperlipidemia and impaired renal function in patients with ORG, indicating that hyperlipidemia causes damage in kidney cells. In podocytes, the endocytosis of lipids triggers an intracellular oxidative stress response that disrupts cellular integrity, resulting in proteinuria and glomerular sclerosis. However, the specific molecular mechanisms through which podocytes endocytose lipids remain unclear. Here, we demonstrated the enhanced endocytosis of lipids by podocytes from patients with ORG. This response was associated with decreased expression of phosphatase and tensin homolog (PTEN). In vitro silencing of PTEN promoted the endocytosis of low-density lipoprotein in mouse podocytes. Conversely, overexpression of PTEN inhibited the endocytosis of lipoproteins in podocytes. PTEN directly dephosphorylates and activates the actin-depolymerizing factor cofilin-1, leading to depolymerization of filamentous actin (F-actin), which is necessary for endocytosis. Notably, inhibition of PTEN resulted in the phosphorylation and inactivation of cofilin-1, leading to F-actin formation that enhanced the endocytosis of lipoproteins in podocytes. When hyperlipidemia was induced in mice with podocyte-specific deletion of PTEN, these mice recapitulated the major pathophysiological features of ORG. Thus, PTEN downregulation in podocytes may contribute to the pathogenesis of ORG.

The signaling axis of Rac1-TFEB regulates autophagy and tumorigenesis.

Macroautophagy (hereafter referred to as autophagy) plays essential roles in cellular and organismal homeostasis. Transcription factor EB (TFEB) is a master regulator of autophagy and lysosome biogenesis. It is not fully understood how the function of TFEB in autophagy pathway is regulated. Here, we show that Rac1 GTPase is a negative modulator of autophagy by targeting TFEB. Mechanistically, Rac1 reduces autophagy flux by repressing the expressing of autophagy genes. Further investigation revealed that under nutrient-rich conditions, mammalian target of rapamycin (mTOR) phosphorylates TFEB to facilitate the interaction between Rac1 and TFEB. Biochemical dissection uncovered that guanosine 5'-triphosphate (GTP)-bound form of Rac1 selectively interacts with phosphorylated TFEB. This inhibitory interaction prevents the dephosphorylation and nucleus translocation of TFEB, which hampers the transcriptional activation of autophagy-related genes. Furthermore, Rac1-TFEB axis appeared to be important for tumorigenesis, as overexpression of dephosphorylated mutant of TFEB was able to delay the tumor growth driven by Rac1 overexpression. Together, this study not only elucidates a previously uncharacterized autophagy regulation mechanism involving Rac1 and TFEB under physiological and pathological conditions but also suggests a strategy to treat cancers that are driven by Rac1 overexpression.

Flavonoid-rich Scabiosa comosa inflorescence extract attenuates CCl4-induced hepatic fibrosis by modulating TGF-ß-induced Smad3 phosphorylation.

Scabiosa comosa inflorescence is a traditional Mongolian medicine in the treatment of liver diseases. In the study, we investigated the anti-fibrotic efficacy of flavonoid-rich Scabiosa comosa inflorescence extract (TF-SC) in a rat model of CCl4-induced hepatic fibrosis and explored its underlying mechanism in vitro and in vivo. Rats (Wistar, Male, weight 200-250 g) were injected intraperitoneally with CCl4 (1:1v/v in peanut oil, 2 mL/kg body weight) to induce liver fibrosis, followed by treatment with TF-SC or vehicle. In addition, transforming growth factor-ß1 (TGF-ß1)-activated hepatic stellate cells (HSCs) were used for measuring Smad3 phosphorylation. We found decrease in liver function and liver fibrosis markers in serums. Also, TF-SC decreased hydroxyproline content and collagen deposition in liver tissues. TF-SC also decreased the expression of α-SMA, collagen I and fibronectin in CCl4-induced hepatic fibrosis rats. Mechanistically, TF-SC attenuated liver fibrosis by selectively inhibiting Smad3 phosphorylation. In TGF-ß1-stimulated HSCs, TF-SC blocked the interaction between Smad3 and TGF-ß type I receptor (TßRI), suppressed subsequent phosphorylation and nuclear translocation of Smad3, and down-regulated the transcription of fibrotic genes. In conclusion, the study demonstrated that TF-SC was an effective therapeutic agent for treatment of hepatic fibrosis, and provided a molecular basis through which TF-SC exerts its anti-fibrotic effects.

Increased Level of c-kit in Semen of Infertile Patients with Varicocele.

PURPOSE: Varicocele is the most common risk factor for male infertility, however, not all males with varicocele experience infertility. In fact, most patients with varicocele have normal spermatogenesis. The molecular mechanism of varicocele-associated infertility is yet to be completely understood. The aim of this study is to assess the association of a number of fertility regulatory factors on varicocele associated infertility and to throw light on the mechanism of varicocele-associated infertility. MATERIALS AND METHODS: Semen from 30 infertile patients with varicocele and 30 fertile men with varicocele were collected. The concentrations of the following factors in seminal plasma were determined by ELISA: follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T), androgen binding protein (ABP), transferrin (Trf), inhibin B (INHB) and stem cell factor (SCF). The expression level of c-kit in seminal precipitate of patients with varicocele was detected by real-time PCR. RESULTS: The concentrations of sexual hormones, FSH, LH and T, had no differences between infertile patients with varicocele and fertile men with varicocele (P > 0.05). Factors secreted by Sertoli cells, ABP, Trf, INHB andSCF, showed no significant differences between the two groups (P > 0.05). Interestingly, the expression of c-kit was significant higher in infertile patients with varicocele than that in fertile men with varicocele (P < 0.01). CONCLUSION: Neither the sexual hormones nor the Sertoli cells was responsible for the infertility induced by varicocele.The aberrant expression of c-kit in infertile patients with varicocele may provide new insight into the mechanism of varicocele-associated infertility.

Aberrant expressions of stem cell factor/c-KIT in rat testis with varicocele.

BACKGROUND/PURPOSE: Varicocele (VC) is considered by the World Health Organization as the main cause of male infertility. Studies have shown that VC can affect spermatogenesis and then result in male infertility. But the exact mechanism by which VC affects spermatogenesis is still unclear. Stem cell factor (SCF) and c-KIT receptor are crucial molecules during spermatogenesis in testis. This study aims to investigate whether SCF/c-KIT signaling is involved in the pathophysiology of VC on spermatogenesis. METHODS: Rat models of VC were built (n = 13), and sham-operated rats were used as controls (n = 8). The seminiferous tubules of the testis were observed with hematoxylin and eosin staining, expression of SCF was analyzed via enzyme-linked immunosorbent assay and Western blot, and expression of c-KIT was assessed with Western blot and immunofluorescence. RESULTS: Compared with controls, the seminiferous epithelium was disorganized and had significantly fewer cells in the testes of rats with VC. Expression of SCF increased in testes of VC rats, while expression of c-KIT was decreased. CONCLUSION: These results suggest that sperm counts in seminiferous epithelium are affected by VC, and the SCF/c-KIT system is aberrantly expressed in VC testis, which could be involved in male infertility caused by VC.

Reduced Glutamate Release in Adult BTBR Mouse Model of Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is a developmental disorder characterized by impairments in social and communication abilities, as well as by restricted and repetitive behaviors. The BTBR T + Itpr3 tf (BTBR) mice have emerged as a well characterized and widely used mouse model of a range of ASD-like phenotype, showing deficiencies in social behaviors and unusual ultrasonic vocalizations as well as increased repetitive self-grooming. However, the inherited neurobiological changes that lead to ASD-like behaviors in these mice are incompletely known and still under active investigation. The aim of this study was to further evaluate the structure and neurotransmitter release of the glutamatergic synapse in BTBR mice. C57BL/6J (B6) mice were used as a control strain because of their high level of sociability. The important results showed that the evoked glutamate release in the cerebral cortex of BTBR mice was significantly lower than in B6 mice. And the level of vesicle docking-related protein Syntaxin-1A was reduced in BTBR mice. However, no significant changes were observed in the number of glutamatergic synapse, level of synaptic proteins, density of dendritic spine and postsynaptic density between BTBR mice and B6 mice. Overall, our results suggest that abnormal vesicular glutamate activity may underlie the ASD relevant pathology in the BTBR mice.

Downregulation of human Wnt3 in gastric cancer suppresses cell proliferation and induces apoptosis.

Aberrant activation of Wnt/ß-catenin signaling pathways is closely involved in the occurrence and progression of several types of human malignancies. However, as a fundamental component in this cascade, Wnt3 has not been well understood for the expression level and pathogenic mechanism in gastric carcinogenesis. Here, this research was undertaken to elucidate the important role of Wnt3 in gastric cancer. Wnt3 expression in gastric carcinomas and their respective normal tissues was examined by immunoblotting and immunohistochemistry. In all cases, Wnt3 expression was significantly elevated in gastric carcinomas compared with normal tissues. Knocking down Wnt3 in MGC-803 gastric cancer cells by small interfering RNAs transfection led to an obvious decrease in both transcript and protein levels. Silence of Wnt3 expression in gastric cancer cells inhibited the expression of ß-catenin and cyclin D1 genes in Wnt/ß-catenin pathway, significantly blocked cellular proliferation, delayed cell cycle, suppressed cell invasion and metastasis, accompanied by a higher apoptosis rate. Together, we conclude that upregulation of Wnt3 plays a crucial role in gastric tumorigenesis by inducing proliferation, migration, and invasion and inhibiting apoptosis of cancer cells, and Wnt3 might be a potential target for the treatment of gastric cancer.