The in vitro mechanism of Vaspinregulates the proliferation and steroidogenesis of rat lutein cells.

OBJECTIVE: Stable luteal cell function is an important prerequisite for reproductive ability and embryonic development. However, luteal insufficiency seriously harms couples who have the desire to have a pregnancy, and the most important thing is that there is no complete solution. In addition, Vaspin has been shown to have regulatory effects on luteal cells, but the complex mechanisms involved have not been fully elucidated. Therefore, this study aimed to explore the effect of Vaspin on rat luteal cells and its mechanism. METHODS: Granulosa lutein cells separated from the ovary of female rats were incubated for 24h with gradient concentrations of Vaspin, and granulosa lutein cells incubated with 0.5% bovine serum albumin were used as controls. The proliferation, apoptosis, angiogenesis, progesterone (P4) and estradiol (E2) were detected by CCK-8, Anneixn-FITC/PI staining, angiogenesis experiment and ELISA. Western blot was applied to observe the expression levels of proteins related to cell proliferation, apoptosis, angiogenesis and MEK/MAPK signaling pathway. RESULTS: Compared with the Control group, Vaspin could significantly up-regulate the proliferation of granulosa lutein cells and reduce the apoptosis. Moreover, Vaspin promoted the angiogenesis of granulosa lutein cells and the production of P4 and E2 in a concentration-dependent manner. Furthermore, Vaspin up-regulated the CyclinD1, CyclinB1, Bcl2, VEGFA and FGF-2 expression in granulosa lutein cells, and down-regulated the level of Bax. Also, Vaspin increased the p-MEK1 and p-p38 levels. CONCLUSION: Vaspin can up-regulate the proliferation and steroidogenesis of rat luteal cells and reduce apoptosis, which may be related to the influence of MEK/MAPK activity.

Genetic analysis of seven patients with inherited ichthyosis and Nagashima­type palmoplantar keratoderma.

Inherited ichthyosis comprises a series of heterogeneous dermal conditions; it mainly manifests as widespread hyperkeratosis, xerosis and scaling of the skin. At times, overlapping symptoms require differential diagnosis between ichthyosis and several other similar disorders. The present study reports seven patients with confirmed or suspected to be associated with ichthyosis by conducting a thorough clinical and genetic investigation. Genetic testing was conducted using whole­exome sequencing, with Sanger sequencing as the validation method. The MEGA7 program was used to analyze the conservation of amino acid residues affected by the detected missense variants. The enrolled patients exhibited ichthyosis­like but distinct clinical manifestations. Genetic analysis identified diagnostic variations in the FLG, STS, KRT10 and SERPINB7 genes and clarified the carrying status of each variant in the respective family members. The two residues affected by the detected missense variants remained conserved across multiple species. Of note, the two variants, namely STS: c.452C>T(p.P151L) and c.647_650del(p.L216fs) are novel. In conclusion, a clear genetic differential diagnosis was made for the enrolled ichthyosis­associated patients; the study findings also extended the mutation spectrum of ichthyosis and provided solid evidence for the counseling of the affected families.

Prognostic Impact of Serum SCC Antigen in the 566 Upfront Surgery Group of Esophageal Squamous Cell Carcinoma: A Multi-Institutional Study of the Japan Esophageal Society.

PURPOSE: This study aimed to determine the clinicopathologic and prognostic significance of squamous cell carcinoma antigen (SCC-Ag) in patients with esophageal SCC who underwent radical surgery without neoadjuvant therapy. METHODS: This study included 566 patients with primary esophageal SCC who underwent radical resection without neoadjuvant therapy at 15 Japanese hospitals between 2008 and 2016. The cutoff value of SCC-Ag was 1.5 ng/mL based on the receiver operating characteristic curves. Preoperative SCC-Ag and postoperative SCC-Ag were analyzed to evaluate clinicopathological and prognostic significance. Survival curves were compared between the SCC-Ag-positive group and the SCC-Ag-negative group. The prognostic impact of SCC-Ag was evaluated using univariate and multivariate analyses. RESULTS: The preoperative SCC-Ag-positive rate was 23.5% (133/566). SCC-Ag-positive status was significantly associated with old age (p = 0.042), tumor depth (p <0.001), and tumor stages (p <0.001). The preoperative SCC-Ag-positive group had significantly poorer overall survival than the SCC-Ag-negative group (p = 0.030), but it was not an independent predictor of poor prognosis. Postoperative SCC-Ag-positive status was an independent risk factor for poor overall survival (p = 0.034). CONCLUSION: Both pre- and postoperative SCC-Ag-positive statuses were significantly associated with poor prognosis. Postoperative SCC-Ag-positive status was an independent risk factor for predicting overall survival.

Combination of Fushengong decoction with Western medicine on patients with chronic renal failure: An observational study.

Chronic renal failure (CRF) causes a reduction in glomerular filtration rate and damage to renal parenchyma. Fushengong decoction (FSGD) showed improvement in renal function in CRF rats. This study aims to analyze the differentially expressed proteins in CRF patients treated with Western medicine alone or in combination with FSGD. Sixty patients with CRF recruited from Yongchuan Traditional Chinese Medicine Hospital affiliated to Chongqing Medical University were randomly assigned into control (treated with Western medicine alone) and observation groups (received additional FSGD treatment thrice daily for 8 weeks). The clinical efficacy and changes in serum Bun, serum creatinine, Cystatin C, and transforming growth factor beta 1 (TGF-ß1) before and after treatment were observed. We employed isotope relative labeling absolute quantification labeling and liquid chromatography-mass spectrometry to identify differentially expressed proteins and carried out bioinformatics Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses. Patients in the observation group showed greater clinical improvement and lower levels of serum Bun, serum creatinine, Cyc-c, and TGF-ß1 than the control group. We identified 32 differentially up-regulated and 52 down-regulated proteins in the observation group. These proteins are involved in the blood coagulation system, protein serine/threonine kinase activity, and TGF-ß, which are closely related to the pathogenesis of CRF. Protein-protein-interaction network analysis indicated that candidate proteins fibronectin 1, fibrinogen alpha chain, vitronectin, and Serpin Family C Member 1 were in the key nodes. This study provided an experimental basis suggesting that FSGD combined with Western medicine could significantly improve renal function and renal fibrosis of CRF patients, which may be through the regulation of fibronectin 1, fibrinogen alpha chain, vitronectin, Serpin Family C Member 1, TGF-ß, and the complement coagulation pathway (see Graphical abstract S1, Supplemental Digital Content, http://links.lww.com/MD/L947).

Clinical Significance of Serum SCC Levels Before Treatment for Locally Advanced Cervical Squamous Cell Carcinoma.

BACKGROUND/AIM: Although serum squamous cell carcinoma (SCC) antigen values are known to be useful in predicting the prognosis of cervical SCC, they have only been examined in a cursory manner. This study aimed to meticulously investigate the clinical significance of serum SCC antigen levels in patients with locally advanced cervical squamous cell carcinoma (LACSC). PATIENTS AND METHODS: The study included patients who were diagnosed with local stage (T-stage) 1b3/2/3 LACSC and underwent initial treatment at our institute between January 2006 and December 2016 (T-1b3: n=30; T-2: n=75; T-3: n=34). The patients were divided into three groups based on pre-treatment SCC values, and differences in clinical background, laboratory and pathology findings, and prognosis were examined. RESULTS: No significant difference in the SCC distribution was observed among the T-1b3/2/3 cases with elevated SCC levels. In stages T-1b3, T-2, and T-3, most recurrences in the SCC-High group were distant (T-1b3: three out of five recurrences; T-2: six out of seven recurrences; T-3: four out of eight recurrences), while most recurrences in the SCC-Low group were pelvic (T-1b3: two out of three recurrences; T-2: eight out of eight recurrences; T-3: three out of three recurrences). CONCLUSION: In LACSC, serum SCC antigen levels before treatment correlate strongly with the recurrence site. Patients with low levels should be closely monitored for local recurrence, whereas those with high levels warrant vigilance for distant recurrence.

Average log change rate of pretreatment squamous cell carcinoma antigen after concurrent chemoradiotherapy in stage IIIC1 cervical squamous cell carcinoma.

We aimed to determine whether pretreatment squamous cell carcinoma antigen (SCC-Ag) levels and the average logarithmic change in SCC-Ag levels ( Δ log SCC-Ag Δ time ) after concurrent chemoradiotherapy (CCRT) could predict treatment outcomes in patients with stage IIIC1 cervical squamous cell carcinoma (SCC). We analyzed 168 patients with stage IIIC1 cervical SCC who underwent primary CCRT and collected data on age, local extension, treatment details, hematological parameters, and tumor markers such as SCC-Ag and carcinoembryonic antigen 21-1 (Cyfra). Predictive performances of pretreatment SCC-Ag levels and Δ log SCC-Ag Δ time were assessed using receiver operating characteristic curves. Survival analysis was performed using the Cox regression model and Kaplan-Meier plots. The combination of pretreatment SCC-Ag levels and Δ log SCC-Ag Δ time showed higher area under the curve values than pretreatment SCC-Ag levels alone (area under the curve; 95% confidence interval [CI] 0.708 [0.581-0.836] vs. 0.666 [0.528-0.804], respectively). Pretreatment SCC-Ag (≥ 5 ng/ml and Cyfra levels (≥ 3.15 ng/ml) and Δ log SCC-Ag Δ time (≥ - 1.575) were significant predictors of disease-specific survival. The 5-year disease-specific survival rates significantly differed among the low-, intermediate-, and high-risk groups. Risk stratification using both pretreatment SCC-Ag levels and Δ log SCC-Ag Δ time may predict treatment outcomes of patients with stage IIIC1 SCC.

Application Value of CYFRA21-1 Combined with NSE, CEA, and SCC-Ag in Lung Cancer.

BACKGROUND: This study aims to investigate the application value of serum cytokeratin 19 fragment (CYFRA21-1) combined with nerve-specific enolase (NSE), carcinoembryonic antigen (CEA), and squamous cell carcinoma antigen (SCC-Ag) in the diagnosis of lung cancer (LC). METHODS: A total of 831 cases of LC, 360 cases of benign lung disease (BLD) and 102 healthy controls, were enrolled. The data were processed using SPSS, GraphPad Prism, and MedCalc software. RESULTS: The tumor marker (TM) levels in the LC and BLD groups were significantly higher than those in the control group; the CYFRA21-1, NSE, and CEA levels in the patients with LC were higher than in those with BLD. In particular, the increase was predominantly observed for the levels of CEA and CYFRA21-1 in adenocarcinoma (LUAD), CYFRA21-1 and SCC-Ag in squamous cell carcinoma (LUSC), and NSE in small cell carcinoma (SCLC). The CYFRA21-1, NSE, and CEA levels were significantly higher in stage IV than in other stages in LC. Univariate binary logistic analysis showed that increased levels of all four TMs were risk factors for BLD and LC. The area under the curve (AUC) of CYFRA21-1 was most effective in distinguishing patients with BLD or LC from the controls and in distinguishing patients with BLD and LC. The AUCs of combined CYFRA21-1, NSE, and CEA were increased to 0.755, 0.922, and 0.783, respectively, with no significant difference with the AUC of the four combined tests. In the histological classification, the best predictors were CEA, for LUAD, CYFRA21-1 for LUSC, and NSE for SCLC. Moreover, the expression levels of CYFRA21-1, NSE, and CEA significantly decreased after each treatment course. CONCLUSIONS: The combined assay of CYFRA21-1, NSE, and CEA addresses the aspects of accuracy, sensitivity, specificity, and economic cost and should be considered as a potential diagnostic test in LC.

Regulation of CNS pathology by Serpina3n/SERPINA3: The knowns and the puzzles.

Neuroinflammation, blood-brain barrier (BBB) dysfunction, neuron and glia injury/death and myelin damage are common central nervous system (CNS) pathologies observed in various neurological diseases and injuries. Serine protease inhibitor (Serpin) clade A member 3n (Serpina3n), and its human orthologue SERPINA3, is an acute-phase inflammatory glycoprotein secreted primarily by the liver into the bloodstream in response to systemic inflammation. Clinically, SERPINA3 is dysregulated in brain cells, cerebrospinal fluid and plasma in various neurological conditions. Although it has been widely accepted that Serpina3n/SERPINA3 is a reliable biomarker of reactive astrocytes in diseased CNS, recent data have challenged this well-cited concept, suggesting instead that oligodendrocytes and neurons are the primary sources of Serpina3n/SERPINA3. The debate continues regarding whether Serpina3n/SERPINA3 induction represents a pathogenic or a protective mechanism. Here, we propose possible interpretations for previously controversial data and present perspectives regarding the potential role of Serpina3n/SERPINA3 in CNS pathologies, including demyelinating disorders where oligodendrocytes are the primary targets. We hypothesise that the 'good' or 'bad' aspects of Serpina3n/SERPINA3 depend on its cellular sources, its subcellular distribution (or mis-localisation) and/or disease/injury types. Furthermore, circulating Serpina3n/SERPINA3 may cross the BBB to impact CNS pathologies. Cell-specific genetic tools are critically important to tease out the potential roles of cell type-dependent Serpina3n in CNS diseases/injuries.

Elevated Kallistatin promotes the occurrence and progression of non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide, and the development of non-alcoholic steatohepatitis (NASH) might cause irreversible hepatic damage. Hyperlipidemia (HLP) is the leading risk factor for NAFLD. This study aims to illuminate the causative contributor and potential mechanism of Kallistatin (KAL) mediating HLP to NAFLD. 221 healthy control and 253 HLP subjects, 62 healthy control and 44 NAFLD subjects were enrolled. The plasma KAL was significantly elevated in HLP subjects, especially in hypertriglyceridemia (HTG) subjects, and positively correlated with liver injury. Further, KAL levels of NAFLD patients were significantly up-regulated. KAL transgenic mice induced hepatic steatosis, inflammation, and fibrosis with time and accelerated inflammation development in high-fat diet (HFD) mice. In contrast, KAL knockout ameliorated steatosis and inflammation in high-fructose diet (HFruD) and methionine and choline-deficient (MCD) diet-induced NAFLD rats. Mechanistically, KAL induced hepatic steatosis and NASH by down-regulating adipose triglyceride lipase (ATGL) and comparative gene identification 58 (CGI-58) by LRP6/Gɑs/PKA/GSK3ß pathway through down-regulating peroxisome proliferator-activated receptor γ (PPARγ) and up-regulating kruppel-like factor four (KLF4), respectively. CGI-58 is bound to NF-κB p65 in the cytoplasm, and diminishing CGI-58 facilitated p65 nuclear translocation and TNFα induction. Meanwhile, hepatic CGI-58-overexpress reverses NASH in KAL transgenic mice. Further, free fatty acids up-regulated KAL against thyroid hormone in hepatocytes. Moreover, Fenofibrate, one triglyceride-lowering drug, could reverse hepatic steatosis by down-regulating KAL. These results demonstrate that elevated KAL plays a crucial role in the development of HLP to NAFLD and may be served as a potential preventive and therapeutic target.

Split Gp41-1 intein splicing as a model to evaluate the cellular location of the oncosuppressor Maspin in an in vitro model of osteosarcoma.

Inteins are proteins involved in the protein splicing mechanism, an autoprocessing event, where sequences (exteins) separated by inteins become ligated each other after recombination. Two kinds of inteins have been described, contiguous inteins and split inteins. The former ones are transcribed and translated as a single peptide along with their exteins, while the latter are fragmented between two different genes and are transcribed and translated separately. The aim of this study is to establish a method to obtain a fluorescent eukaryotic protein to analyze its cellular localization, using the natural split gp41-1 inteins. We chose natural split inteins due to their distribution in all three domains of life. Two constructs were prepared, one containing the N-terminal split intein along with the N-moiety of the Red Fluorescent Protein (RFP) and a second construct containing the C-terminal of split intein, the C-moiety of RFP and the gene coding for Maspin, a tumor suppressor protein. The trans-splicing was verified by transfecting both N-terminal and C-terminal constructs into mammalian cells. The success of the recombination event was highlighted through the fluorescence produced by reconstituted RFP after recombination, along with the overlap of the red fluorescence produced by recombined RFP and the green fluorescence produced by the hybridization of the recombinant Maspin with a specific antibody. In conclusion, we opted to use this mechanism of recombination to obtain a fluorescent Maspin instead to express a large fusion protein, considering that it could interfere with Maspin's structure and function.

Smoking behavior associated upregulation of SERPINB12 promotes proliferation and metastasis via activating WNT signaling in NSCLC.

BACKGROUND: Non-small cell lung cancer (NSCLC) is the leading cause of morality among all malignant tumors. Smoking is one of the most important causes of NSCLC, which contributes not only to the initiation of NSCLC but also to its progression. The identification of specific biomarkers associated with smoking will promote diagnosis and treatment. METHODS: Data mining was used to identify the smoking associated gene SERPINB12. CCK8 assays, colony formation assays, a mouse xenograft model and transwell assays were performed to measure the biological functions of SERPINB12 in NSCLC. GSEA, luciferase reporter assays and immunofluorescence were conducted to explore the potential molecular mechanisms of SERPINB12 in NSCLC. RESULTS: In this study, by data mining the TCGA database, we found that SERPINB12 was greatly upregulated in NSCLC patients with cigarette consumption behavior, while the expression level was positively correlated with disease grade and poor prognosis. SERPINB12 is a kind of serpin peptidase inhibitor, but its function in malignant tumors remains largely unknown. Functionally, knockdown of SERPINB12 observably inhibited the proliferation and metastasis of NSCLC cells in vitro and in vivo. Moreover, downregulation of SERPINB12 attenuated Wnt signaling by inhibiting the nuclear translocation of ß-catenin, which explained the molecular mechanism underlying tumor progression. CONCLUSIONS: In conclusion, SERPINB12 functions as a tumorigenesis factor, which could be a promising biomarker for NSCLC patients with smoking behavior, as well as a therapeutic target.

The role of adipokines in osteoporosis management: a mini review.

The prevalence of osteoporosis has been on the rise globally. With ageing populations, research has sought therapeutic solutions in novel areas. One such area is that of the adipokines. Current literature points to an important role for these chemical mediators in relation to bone metabolism. Well-established adipokines have been broadly reported upon. These include adiponectin and leptin. However, other novel adipokines such as visfatin, nesfatin-1, meteorin-like protein (Metrnl), apelin and lipocalin-2 are starting to be addressed pre-clinically and clinically. Adipokines hold pro-inflammatory and anti-inflammatory properties that influence the pathophysiology of various bone diseases. Omentin-1 and vaspin, two novel adipokines, share cardioprotective effects and play essential roles in bone metabolism. Studies have reported bone-protective effects of omentin-1, whilst others report negative associations between omentin-1 and bone mineral density. Lipocalin-2 is linked to poor bone microarchitecture in mice and is even suggested to mediate osteoporosis development from prolonged disuse. Nesfatin-1, an anorexigenic adipokine, has been known to preserve bone density. Animal studies have demonstrated that nesfatin-1 treatment limits bone loss and increases bone strength, suggesting exogenous use as a potential treatment for osteopenic disorders. Pre-clinical studies have shown adipokine apelin to have a role in bone metabolism, mediated by the enhancement of osteoblast genesis and the inhibition of programmed cell death. Although many investigations have reported conflicting findings, sufficient literature supports the notion that adipokines have a significant influence on the metabolism of bone. This review aims at highlighting the role of novel adipokines in osteoporosis while also discussing their potential for treating osteoporosis.

Pigment epithelium­derived factor inhibits proliferation, invasion and angiogenesis, and induces ferroptosis of extravillous trophoblasts by targeting Wnt­ß­catenin/VEGF signaling in placenta accreta spectrum.

Placenta accreta spectrum (PAS) is one of the most dangerous complications in obstetrics, which can lead to severe postpartum bleeding and shock, and even necessitate uterine removal. The abnormal migration and invasion of extravillous trophoblast cells (EVTs) and enhanced neovascularization occurring in an uncontrolled manner in time and space are closely related to the abnormal expression of pro­angiogenic and anti­angiogenic factors. The pigment epithelium­derived factor (PEDF) is a multifunctional regulatory factor that participates in several important biological processes and is recognized as the most efficient inhibitor of angiogenesis. The present study aimed to explore the effects of PEDF on EVT phenotypes and the underlying mechanisms in PAS. HTR­8/SVneo cells were transfected to overexpress or knock down PEDF. Cell proliferation and invasion were assessed using Cell Counting Kit­8, 5­ethynyl­2'­deoxyuridine and Transwell assays. In vitro angiogenesis was analyzed using tube formation assays. The degree of ferroptosis was assessed by evaluating the levels of lipid reactive oxygen species, total iron, Fe2+, malondialdehyde and reduced glutathione using commercial kits. The expression levels of biomarkers of ferroptosis, angiogenesis, cell proliferation and Wnt signaling were examined by western blotting. PEDF overexpression decreased the proliferation, invasion and angiogenesis, and induced ferroptosis of EVTs. Activation of Wnt signaling with BML­284 and overexpression of vascular endothelial growth factor (VEGF) reversed the PEDF overexpression­induced suppression of cell proliferation, invasion and tube formation. PEDF overexpression­induced ferroptosis was also decreased by Wnt agonist treatment and VEGF overexpression. It was predicted that PEDF suppressed the proliferation, invasion and angiogenesis, and increased ferroptosis in EVTs by decreasing Wnt­ß­catenin/VEGF signaling. The findings of the present study suggested a novel regulatory mechanism of the phenotypes of EVTs and PAS.

Identification and functional analysis of a serine protease inhibitor using machine learning strategy.

In the intricate realm of animal biology, a multitude of vital processes heavily rely on precisely orchestrated proteinase cascades, but the potential for havoc makes proteinase inhibitors indispensable, with serine proteinase inhibitors (serpins) at the forefront, serving as custodians of homeostasis and participating in various critical biological processes. Importantly, there are still many unexplored facets of serpin functionality. In this study, we focused on the serpin family proteins from Marsupenaeus japonicus, utilizing a fine-tuned pretrained protein language model. This approach led to the identification and evolutionary validation of 28 serpins, one of which, referred to as Mjserpin-1, was both computationally and experimentally demonstrated to show potential as an antiviral and apoptosis inhibitor. Our research unveils exciting prospects for the fusion of state-of-the-art artificial intelligence and rich bioinformatics, holding the promise of significant discoveries that could pave the way for future therapeutic advancements.

Role of human Kallistatin in glucose and energy homeostasis in mice.

OBJECTIVE: Kallistatin (KST), also known as SERPIN A4, is a circulating, broadly acting human plasma protein with pleiotropic properties. Clinical studies in humans revealed reduced KST levels in obesity. The exact role of KST in glucose and energy homeostasis in the setting of insulin resistance and type 2 diabetes is currently unknown. METHODS: Kallistatin mRNA expression in human subcutaneous white adipose tissue (sWAT) of 47 people with overweight to obesity of the clinical trial "Comparison of Low Fat and Low Carbohydrate Diets With Respect to Weight Loss and Metabolic Effects (B-SMART)" was measured. Moreover, we studied transgenic mice systemically overexpressing human KST (hKST-TG) and wild type littermate control mice (WT) under normal chow (NCD) and high-fat diet (HFD) conditions. RESULTS: In sWAT of people with overweight to obesity, KST mRNA increased after diet-induced weight loss. On NCD, we did not observe differences between hKST-TG and WT mice. Under HFD conditions, body weight, body fat and liver fat content did not differ between genotypes. Yet, during intraperitoneal glucose tolerance tests (ipGTT) insulin excursions and HOMA-IR were lower in hKST-TG (4.42 ± 0.87 AU, WT vs. 2.20 ± 0.27 AU, hKST-TG, p < 0.05). Hyperinsulinemic euglycemic clamp studies with tracer-labeled glucose infusion confirmed improved insulin sensitivity by higher glucose infusion rates in hKST-TG mice (31.5 ± 1.78 mg/kg/min, hKST-TG vs. 18.1 ± 1.67 mg/kg/min, WT, p < 0.05). Improved insulin sensitivity was driven by reduced hepatic insulin resistance (clamp hepatic glucose output: 7.7 ± 1.9 mg/kg/min, hKST-TG vs 12.2 ± 0.8 mg/kg/min, WT, p < 0.05), providing evidence for direct insulin sensitizing effects of KST for the first time. Insulin sensitivity was differentially affected in skeletal muscle and adipose tissue. Mechanistically, we observed reduced Wnt signaling in the liver but not in skeletal muscle, which may explain the effect. CONCLUSIONS: KST expression increases after weight loss in sWAT from people with obesity. Furthermore, human KST ameliorates diet-induced hepatic insulin resistance in mice, while differentially affecting skeletal muscle and adipose tissue insulin sensitivity. Thus, KST may be an interesting, yet challenging, therapeutic target for patients with obesity and insulin resistance.

PEDF-derived peptide protects against Amyloid-ß toxicity in vitro and prevents retinal dysfunction in rats.

Amyloid-beta (Aß), a family of aggregation-prone and neurotoxic peptides, has been implicated in the pathophysiology of age-related macular degeneration (AMD). We have previously shown that oligomeric and fibrillar species of Aß42 exerted retinal toxicity in rats, but while the consequences of exposure to amyloid were related to intracellular effects, the mechanism of Aß42 internalization in the retina is not well characterized. In the brain, the 67 kDa laminin receptor (67LR) participates in Aß-related neuronal cell death. A short peptide derived from pigment epithelium-derived factor (PEDF), formerly designated PEDF-335, was found to mitigate experimental models of ischemic retinopathy via targeting of 67LR. In the present study, we hypothesized that 67LR mediates the uptake of pathogenic Aß42 assemblies in the retina, and that targeting of this receptor by PEDF-335 may limit the internalization of Aß, thereby ameliorating its retinotoxicity. To test this assumption ARPE-19 cells in culture were incubated with PEDF-335 before treatment with fibrillar or oligomeric structures of Aß42. Immunostaining confirmed that PEDF-335 treatment substantially prevented amyloid internalization into ARPE-19 cells and maintained their viability in the presence of toxic oligomeric and fibrillar Aß42 entities in vitro. FRET competition assay was performed and confirmed the binding of PEDF-335 to 67LR in RPE-like cells. Wild-type rats were treated with intravitreal PEDF-335 in the experimental eye 2 days prior to administration of retinotoxic Aß42 oligomers or fibrils to both eyes. Retinal function was assessed by electroretinography through 6 weeks post injection. The ERG responses in rats treated with oligomeric or fibrillar Aß42 assemblies were near-normal in eyes previously treated with intravitreal PEDF-335, whereas those measured in the control eyes treated with injection of the Aß42 assemblies alone showed pathologic attenuation of the retinal function through 6 weeks. The retinal presence of 67LR was determined ex vivo by immunostaining and western blotting. Retinal staining demonstrated the constitutional expression of 67LR mainly in the retinal nuclear layers. In the presence of Aß42, the levels of 67LR were increased, although its retinal distribution remained largely unaltered. In contrast, no apparent differences in the retinal expression level of 67LR were noted following exposure to PEDF-335 alone, and its pattern of localization in the retina remained similarly concentrated primarily in the inner and outer nuclear layers. In summary, we found that PEDF-335 confers protection against Aß42-mediated retinal toxicity, with significant effects noted in cells as well as in vivo in rats. The effects of PEDF-335 in the retina are potentially mediated via binding to 67LR and by at least partial inhibition of Aß42 internalization. These results suggest that PEDF-335 may merit further consideration in the development of targeted inhibition of amyloid-related toxicity in the retina. More broadly, our observations provide evidence on the importance of extracellular versus intracellular Aß42 in the retina and suggest concepts on the molecular mechanism of Aß retinal pathogenicity.

Gehua Jiejiu Dizhi decoction ameliorates alcoholic fatty liver in mice by regulating lipid and bile acid metabolism and with exertion of antioxidant stress based on 4DLabel-free quantitative proteomic study.

OBJECTIVE: To analyze the effect and molecular mechanism of Gehua Jiejiu Dizhi decoction (, GJDD) on alcoholic fatty live disease (AFLD) by using proteomic methods. METHODS: The male C57BL/6J mouse were randomly divided into four groups: control group, model group, GJDD group and resveratrol group. After the AFLD model was successfully prepared by intragastric administration of alcohol once on the basis of the Lieber-DeCarli classical method, the GJDD group and resveratrol group were intragastrically administered with GJDD (4900 mg/kg) and resveratrol (400 mg/kg) respectively, once a day for 9 d. The fat deposition of liver tissue was observed and evaluated by oil red O (ORO) staining. 4DLabel-free quantitative proteome method was used to determine and quantify the protein expression in liver tissue of each experimental group. The differentially expressed proteins were screened according to protein expression differential multiples, and then analyzed by Gene ontology classification and Kyoto Encyclopedia of Genes and Genomes pathway enrichment. Finally, expression validation of the differentially co-expressed proteins from control group, model group and GJDD group were verified by targeted proteomics quantification techniques. RESULTS: In semiquantitative analyses of ORO, all kinds of steatosis (ToS, MaS, and MiS) were evaluated higher in AFLD mice compared to those in GJDD or resveratrol-treated mice. 4DLabel-free proteomics analysis results showed that a total of 4513 proteins were identified, of which 3763 proteins were quantified and 946 differentially expressed proteins were screened. Compared with the control group, 145 proteins were up-regulated and 148 proteins were down-regulated in the liver tissue of model group. In addition, compared with the model group, 92 proteins were up-regulated and 135 proteins were down-regulated in the liver tissue of the GJDD group. 15 differentially co-expressed proteins were found between every two groups (model group vs control group, GJDD group vs model group and GJDD group vs control group), which were involved in many biological processes. Among them, 11 differentially co-expressed key proteins (Aox3, H1-5, Fabp5, Ces3a, Nudt7, Serpinb1a, Fkbp11, Rpl22l1, Keg1, Acss2 and Slco1a1) were further identified by targeted proteomic quantitative technology and their expression patterns were consistent with the results of 4D label-free proteomic analysis. CONCLUSIONS: Our study provided proteomics-based evidence that GJDD alleviated AFLD by modulating liver protein expression, likely through the modulation of lipid metabolism, bile acid metabolism and with exertion of antioxidant stress.

Kallistatin leads to cognition impairment via downregulating glutamine synthetase.

In many neurodegenerative disorders, such as Alzheimer's disease (AD), glutamate-mediated neuronal excitotoxicity is considered the basis for cognitive impairment. The mRNA and protein expression of SERPINA4(Kallistatin) are higher in patients with AD. However, whether Kallistatin plays a regulatory role in glutamate-glutamine cycle homeostasis remains unclear. In this study, we identified impaired cognitive function in Kallistatin transgenic (KAL-TG) mice. Baseline glutamate levels were elevated and miniature excitatory postsynaptic current (mEPSC) frequency was increased in the hippocampus, suggesting the impairment of glutamate homeostasis in KAL-TG mice. Mechanistically, we demonstrated that Kallistatin promoted lysine acetylation and ubiquitination of glutamine synthetase (GS) and facilitated its degradation via the proteasome pathway, thereby downregulating GS. Fenofibrate improved cognitive memory in KAL-TG mice by downregulating serum Kallistatin. Collectively, our study findings provide insights the mechanism by which Kallistatin regulates cognitive impairment, and suggest the potential of fenofibrate to prevente and treat of AD patients with high levels of Kallistatin.

Synergy between PEDF and Doxorubicin in Breast Cancer Cells: Effects on Metastatic and Metabolic Pathways.

Pigment epithelium-derived factor (PEDF), a serine protease inhibitor (Serpin) family member, shows promise in inhibiting tumour growth. In our study, we explored the effects of PEDF on the efficacy of the frontline chemotherapy agent doxorubicin (Dox) in BC cells. We found that Dox+PEDF treatment significantly reduced glucose uptake in MDA-MB-231 cells compared to the control (p = 0.0005), PEDF (p = 0.0137), and Dox (p = 0.0171) alone but paradoxically increased it in MCF-7 cells. Our findings further revealed that PEDF, Dox, and Dox+PEDF substantially hindered tumour cell migration from tumour spheroids, with Dox+PEDF showing the most significant impact (p < 0.0001). We also observed notable decreases in the expression of metastatic markers (uPAR, uPA, CXCR4, MT1-MMP, TNF-α) across all treatment groups (p < 0.0001) in both cell lines. When it comes to metabolic pathways, PEDF increased phosphorylated IRS-1 (p-IRS1) levels in MDA-MB-231 and MCF-7 (p < 0.0001), while Dox decreased it, and the combination led to an increase. In MDA-MB-231 cells, treatment with PEDF, Dox, and the combination led to a notable decrease in both phosphorylated AKT (p-AKT) and total AKT levels. In MCF-7, while PEDF, Dox, and their combination led to a reduction in p-AKT, total levels of AKT increased in the presence of Dox and Dox+PEDF. Combining PEDF with Dox enhances the targeting of metastatic and metabolic pathways in breast cancer cell lines. This synergy, marked by PEDF's increasing roles in cancer control, may pave the way for more effective cancer treatments.