Immunoblotting Methods Research Articles

TNF-α Regulated Bidirectional Interaction Between Bone Marrow Mesenchymal Stem Cells and Articular Chondrocytes.

Articular chondrocytes (ACs) secrete a variety of extracellular matrix components to maintain the functions of articular cartilage. Degeneration of ACs leads to the degeneration of articular cartilage and consequently to osteoarthritis. The secretion of bone marrow mesenchymal stem cells (BMSCs) is capable of protecting ACs from degeneration, and thus BMSCs are widely applied to treat osteoarthritis. This study aims to explore whether BMSCs and ACs will affect the functions of each other through their secretions in the context of osteoarthritis. BMSCs and ACs isolated from rabbits were identified using flow cytometry and immunocytochemistry. Conditioned medium of BMSCs and ACs treated with 0, 5, 10, 20, and 40 ng/ml of tumor necrosis factor-alpha (TNF-α) were collected and used to treat ACs and BMSCs, respectively. The viabilities of ACs and BMSCs treated with condition medium were assessed using a Cell Count Kit-8 (CCK-8) kit. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunoblotting, and enzyme-linked immunosorbent assay (ELISA) methods were employed to evaluate the relative expression levels of genes and proteins, as well as the cytokine concentrations in the supernatant. Immunofluorescence and flow cytometry results indicated that the purity of isolated cells exceeded 95%. CCK-8 analysis showed that 6 hours of treatment with a conditioned medium did not affect the viability of BMSCs and ACs. However, treatment for 12 hours or longer significantly increased the viability of BMSCs (p < 0.05) and significantly decreased the viability of ACs (p < 0.01). RT-qPCR results demonstrated that the relative expression levels of Runx2 (1.15-3.91), Alp (1.06-2.84), TNF (BMSCs: 0.94-2.54; ACs: 1.03-2.64), IL6 (BMSCs: 0.98-2.78; ACs: 0.96-3.71), IL17A (BMSCs: 1.08-5.91; ACs: 0.90-4.20), and IL10 (BMSCs: 0.93-2.82; ACs: 0.89-2.25) genes in conditioned medium-treated BMSCs and ACs were dose-dependently elevated (p < 0.001) by TNF-α treatment. Immunoblotting analysis revealed that the expression levels of RUNX2 (0.53-0.86) and ALP (0.49-0.85) proteins were also dose-dependently elevated (p < 0.001) by TNF-α treatment. ELISA results showed similar TNF-α dose-dependent increases (p < 0.001) in the supernatant concentrations of pro-inflammatory cytokines TNF-α (BMSCs: 36.90 ± 0.75 to 199.38 pg/ml; ACs: 29.76 to 293.99 pg/ml), interleukin (IL)-6 (BMSCs: 4.96-48.24 pg/ml; ACs: 6.12-38.15 pg/ml), IL-17 (BMSCs: 3.06-28.99 pg/ml; ACs: 3.08-28.51 pg/ml), as well as the anti-inflammatory cytokine IL-10 (BMSCs: 6.34-65.02 pg/ml; ACs: 5.30-34.85 pg/ml). Together, these results indicate a TNF-α-regulated bidirectional interaction between BMSCs and ACs, deepening our understanding of the pathogenesis of osteoarthritis and aiding in its prevention and treatment.

Using Immunoblotting for Detection of a Circadian Clock Component, Os-GI, in Rice Leaves.

The adaptability of plants to given environmental conditions is achieved through the regulation of various genes. The functional regulation of genes depends on precise transcriptional and translational controls that, at the end, determine the expression of the translated products (proteins). Here, I describe the immunoblotting method for the detection of proteins expressed in rice leaves. This method includes protein extraction, protein separation, electroblotting, and immune detection.

HTLV infection in urban population from Mato Grosso do Sul, Central Brazil.

Brazil has the highest number of HTLV-1 infection in Latin America, with around one million cases spread unevenly across regions. However, there is a limited number of studies on this infection in the general population. This cross-sectional study aimed to estimate the prevalence of HTLV as well as identify types, and subtypes of HTLV among the urban population of Campo Grande, capital of Mato Grosso do Sul state (MS). Between July 2023 and March 2024, all information was obtained from self-reported interviews, and blood samples were collected and screened for anti-HTLV-1/2 by immunoassay and confirmed using the immunoblot method. The proviral DNA of HTLV-1/2 in positive samples was quantified by real-time PCR (qPCR) and genotyped by nucleotide sequencing (Sanger's method). The study enrolled 611 participants, with the majority being women (90.54%), mixed race (46.32%), heterosexual (87.64%), and with a median age of 39years. The prevalence rate of anti-HTLV-1 infection was 0.82% (CI 95% 0.34-1.96). All positive samples (n = 5) were identified as belonging to the Cosmopolitan subtype, one belonging to Japanese and four to Transcontinental subgroups. Among the five positive individuals, two presented symptoms associated with HTLV-1 infection. This study highlights an intermediate prevalence of HTLV-1 in the urban population of Campo Grande, Mato Grosso do Sul, and provides epidemiological information that could help bridge the gaps in the distribution of HTLV in the general population. Also, medical care was provided for individuals presenting clinical manifestations who were previously unaware of their serological status.

Diagnostic performance of Leishmania braziliensis and Leishmania peruviana antigens in the immunoblot method for the detection of american tegumentary leishmaniasis.

Motivation for the study. To contribute to the immunogenic character of soluble and excretion/secretion antigens of Leishmania braziliensis and Leishmania peruviana with the aim of identifying proteins with diagnostic potential. Main findings. The soluble antigen of Leishmania braziliensis has a sensitivity in the detection of ATL of 87.7%, specificity of 100% and a false positive rate of 20% against sera from patients with Chagas disease and 8.3% with mycosis. Implications. Immunoblot can improve the resolution capacity in the serological diagnosis of American tegumentary Leishmaniasis, particularly in patients where the length of the disease and the clinical form make difficult the diagnosis by parasitological methods. This study aimed to determine the performance of Leishmania braziliensis and Leishmania peruviana antigens in the detection of ATL by using serum samples obtained between 2013 - 2016. The obtained soluble and excretion/secretion antigens were transferred to membrane nitrocellulose by immunoblot assay. The evaluation was carried out against sera confirmed for ATL, at a confidence level of 95%, determining that the soluble antigen of Leishmania braziliensis had a sensitivity of 87.7%, specificity of 100% and area under the curve of 0.95; on the other hand, Leishmania peruviana showed values of 92.3%, 95.7% and 0.94, respectively. According to the results, we recommend that the reported immunogenic regions should be characterized and analyzed in order to continue with the development of recombinant and synthetic proteins, aimed at improving the efficiency of the serological diagnosis of the disease.

WITHDRAWN: Shenmai Injection Exerts the Cardioprotective Effects by Modulating Autophagy-apoptosis via Beclin-1

Background and aimDespite the excellent efficacy of anthracyclines, their clinical application is subject to cardiotoxicity. Studies have shown that Shenmai Injection(SMI) alleviated anthracycline-induced myocardial injury, however, the exact mechanism has not been clarified. Experimental procedureEach intervention was explored by detecting the viability of cardiomyocytes, LDH leakage rate, and CK levels. Finally, the effects of each intervention on autophagy-apoptosis and Beclin-1 were observed by using mCherry-EGFP-LC3B double fluorescence method, Annexin V/propidium iodide (PI), and protein immunoblotting method to explain the mechanism of Shenmai Injection. Key resultsThe viability of H9c2 cells in the model group decreased, accompanied by elevated LDH leakage rate and CK levels after doxorubicin intervention (P<0.01 or P<0.05). In contrast, SMI reversed above situation (P<0.01 or P<0.05). Besides, autophagy and apoptosis up-regulated in the model group (P<0.01), accompanied by upregulated Beclin-1, LC3-II, LC3-II/LC3-I, apoptosis effector proteins Cleaved-Caspase-9, Cleaved-Caspase-9/Caspase-9, Cleaved-Caspase-3, Cleaved-Caspase-3/Caspase-3 expressions and the downregulation of p62 protein, anti-apoptotic protein Bcl2 expressions (P<0.01 or P<0.05). Nonetheless, autophagy and apoptosis were decreased with the help of SMI (P<0.01 or P<0.05). ConclusionSMI alleviated autophagy and apoptosis of damaged cardiomyocytes by regulating Beclin-1.

Serological Assessment of Lyme borreliosis in Bulgaria: A Nationwide Study.

Lyme borreliosis (LB), a tick-borne infection caused by bacteria in the Borrelia burgdorferi sensu lato complex, is increasingly prevalent on the Balkan Peninsula, including Bulgaria, where it is the most common tick-borne disease. This study aimed to assess the seroprevalence of LB across Bulgaria by analyzing 1892 serum samples for specific IgG antibodies using a two-tier testing protocol involving an ELISA and immunoblot methods. The results revealed an overall seroprevalence rate of 5.4%, with significant variation based on age, sex, and residence. Seroprevalence increased with age, peaking at 8.4% in individuals over 65 years. Males had a seroprevalence of 8.4% compared to 3.3% in females, and rural residents showed higher seroprevalence (10.2%) compared to urban residents (4.4%). Regional analysis indicated that seroprevalence ranged from 0.0% to 20.0%, with higher rates in northern provinces such as Gabrovo (18.9%) and Targovishte (20.0%). This study highlights the importance of two-step testing protocols for accurate diagnosis and underscores the need for increased awareness and further research to enhance public health measures and the management of LB in Bulgaria.

Prevalence of Allergen-Specific Immunoglobulin E in 57558 Patients in 2012-2022

The study aims to preliminarily investigate the prevalence characteristics of allergen-specific immunoglobulin E (IgE) in 57558 patients over the past decade by examining its distribution in the province and exploring its associations with age, sex, temperature, and relative humidity, providing insights for the prevention and diagnosis of allergic diseases in the Sichuan region. A retrospective analysis was conducted on a cohort of 57558 patients who underwent allergen testing (by means of EUROIMMUN immunoblotting method) at West China Hospital, Sichuan University between August 2012 and February 2022. The clinical data of these patients were collected to establish a comprehensive database, while the temperature and humidity records of the corresponding timeframe were gathered for further analysis. The positive results from the allergen tests were categorized into four levels, including weakly positive (±), positive (+), moderately positive (++), and strongly positive (+++). Statistical analyses were performed using SPSS 25.0, with Chi-square tests conducted to compare count data and Pearson's correlation tests done conducted to assess the relationships between different types of allergens and temperature/relative humidity. P<0.05 was applied to determine statistically significant differences. GraphPad Prism 9.0.0 was utilized to generate visual representations of the data. The overall positivity rate of allergen-specific IgE among the 57558 samples was 30.69%. The top five allergens that elicited positive results were dust mite mix 1 (14.46%), crab (6.67%), soybean (4.72%), fish mix 1 (4.64%), and cockroach (4.34%). Notably, weakly positive (±) results were predominant for allergens such as eggs, peanuts, soybeans, cow's milk, beef, mutton, crab, shrimp, fish mix 1, cockroach, humulus japonicus, ambrosia artemisifolia, artemisia vulgaris, tree mix 2, house dust, and mold mix 1, collectively constituting over 40% of the positive outcomes. In contrast, cat hair and dog dander exhibited an equal distribution of approximately 25% for each positive levels, while mite mix 1 demonstrated the highest proportion of strongly positive results (+++), accounting for 37.66% of all positive results. Sex disparities in positivity rates were evident for various allergens, with significant differences observed for peanut, soybean, crab, shrimp, fish mix 1, cockroach, ambrosia artemisifolia, tree mix 2, cat hair, dog dander, and mite mix 1. Furthermore, the study identified age-related trends in allergen positivity rates, with a general decline observed across most allergens with increasing age. The positive rate of at least one food allergen was highest in the 0-10 age group (36.18%), and the positive rate of at least one inhalation allergen was highest in the 11-20 age group (45.35%). Noteworthy correlations were observed between allergen-specific IgE positivity and environmental factors, including a strong negative correlation between cow's milk allergy and relative humidity ( r=-0.640, P<0.05), a strong negative correlation of artemisia vulgaris sensitivity with temperature ( r Mean high temperature=-0.695, r Mean low temperature=-0.692, P<0.05), and a very strong positive correlation of mold mix 1 sensitivity with relative humidity ( r=0.704, P<0.05). Allergen-specific IgE positivity is associated with genetic factors, demonstrates significant sex- and age-related characteristics in the population, and is influenced by changes in local temperature and relative humidity.

Real-world performance of HIV low viral load values in diagnosing acute HIV infection in a tertiary care hospital in Beijing, China

BackgroundEarly diagnosis of HIV infection decreases the time from HIV diagnosis to viral suppression and reduces further HIV transmission. The Chinese Guidelines for the Diagnosis and Treatment of HIV/AIDS (2021 edition) state that an HIV RNA level > 5,000 copies/mL is the threshold for diagnosing HIV infection. The impact of low viral load values on HIV diagnosis needs to be investigated.MethodsThere were 3455 human immunodeficiency virus (HIV1 + 2) antibody results (immunoblotting method) and 65,129 HIV viral load values at Beijing Youan Hospital from 2019 to 2022. A total of 2434 patients had both antibody confirmatory results and viral load results. The confirmatory antibody results and HIV viral load results of 2434 patients were analyzed to investigate the impact of low viral load values on HIV diagnosis.ResultsOf the 2434 patients who had both confirmatory antibody results and viral load results, the viral load values of 140 patients (5.8%) had viral loads ranging from 40 copies/mL to 5,000 copies/mL before positive confirmatory antibody result, and of these 140 patients, the sample receipt time for the viral load tests of 96 (66.7%) individuals was 1 to 6 days earlier than the corresponding sample receipt time for the confirmatory antibody test. In addition, 34 patients (1.4%) had low viral loads ranging from 40 copies/mL to 1,000 copies/mL before positive confirmatory antibody result.ConclusionThis study revealed that there is a risk of missed diagnosis if a threshold of 5000 copies/mL is used for the diagnosis of HIV infection. These data provide valuable information for the early diagnosis of HIV infection, and our findings have potential benefits for decreasing HIV transmission.

Abstract LB135: Effects of acute psychological stress on DNA damage levels in peripheral blood mononuclear cells: An experimental study with healthy human volunteers

Abstract Increased levels of DNA damage in human peripheral blood mononuclear cells (PBMC), as indicated by classic alkaline comet and γ-H2AX assays, have been widely used in toxicology and epidemiology studies of potential environmental carcinogens. To date, however, little research has explored the possibility that exposure to acute psychological stresses in daily life may acutely increase levels of DNA damage and thus, much like repeated tobacco use, may cumulatively contribute to lifetime cancer risk. The purpose of the present experimental study was to provide a first critical test of the hypothesis that exposure of healthy humans to a brief psychological stress results in increased levels of DNA damage. We also tested preventative effects of a beta-adrenergic receptor blocker (propranolol), a mechanistic pathway for psychological stress effects on DNA supported by preclinical research. Healthy volunteers were recruited by multiple strategies including targeted outreach (telephone/email), with oversampling for age, sex, and minority status. Eligibility was confirmed by self-report, clinical testing (blood &amp; urine), and MD review. On the experimental day, participants were randomized to receive either a single dose of propranolol (80mg) or a placebo. After a 60 min rest period with video distraction, all participants (n=123; 48.8% ≥ 40yr; 52% women; 52% minority) underwent a well-established, 15-minute stress challenge, a speech task and mental arithmetic (Trier Social Stress Test, TSST), with psychological stress confirmed by visual analog scales. Blood samples were collected (via IV line) immediately before and after the TSST. As the primary study outcome, DNA damage in PBMC was assessed (blind) with Comet IV software (% tail intensity) following single cell gel electrophoresis under alkaline conditions with and without in vitro exposure to 25uM H2O2 (comet assay). For a subset of participants with sufficient frozen PBMC samples available (n=43), levels of pan-nuclear γ-H2AX were determined with a validated, sensitive, immunoblot method that detects H2AX phosphorylation at Ser139, and expresses γ-H2AX as the ratio of p-Ser139-H2AX to total H2AX. Exploratory nonparametric analyses revealed that undergoing the TSST significantly increased levels of DNA damage in PBMC (H2O2 comet assay [p&amp;lt;.001] and γ-H2AX biomarker [p=.03], Wilcoxon tests) in the group of participants that was randomized to pretreatment with placebo. In the group pretreated with propranolol before the TSST, a significant increase in γ-H2AX was not found. Conclusion. Supporting the study hypothesis that acute stress can increase levels of DNA damage, the results of the present study highlight the potential importance of additional research to address the possibility that repeated exposures to acute life stresses may cumulatively contribute to increased lifetime risk of cancer. Citation Format: Dana H. Bovbjerg, John Schmitz, Kurt D. Ackerman, Scott R. Beach, Albert D. Donnenberg, Jessica Manculich, Matthew F. Muldoon, Patricia L. Opresko, Patrick M. Tarwater, Hong Wang, Frank J. Jenkins. Effects of acute psychological stress on DNA damage levels in peripheral blood mononuclear cells: An experimental study with healthy human volunteers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB135.

Assessment of the Adsorption Activity of Bacterial Cells Using Yersinia pseudotuberculosis Model

Currently, the Russian Anti-Plague Institute “Microbe” produces diagnostic immunoglobulins used in laboratory diagnostics of plague pathogen. One of the important stages in obtaining this category of drugs is the process of adsorption and removal of cross-reacting antibodies to increase the specificity of the drug. For this purpose, inactivated cells of Yersinia pseudotuberculosis strains are used.The aim of the work was to assess the possibility of using immunochemical methods to analyze the adsorption properties of bacterial cells, followed by an assessment of the impact of cultivation method and conditions, and the variant of the strain used on the adsorption properties of bacterial cells.Materials and methods. Cultivation was carried out on solid and liquid nutrient media; cells were inactivated by temperature and chemical exposure. Adsorption activity was assessed using immunoblotting and inhibitory enzyme-linked immunosorbent assay. Statistical processing of the results was performed using two-factor analysis of variance.Results and discussion. The suitability of immunoblotting and inhibitory ELISA methods for qualitative and quantitative assessment of the adsorption properties of bacterial cells has been demonstrated. It has been established that the adsorption properties of bacterial cells are influenced by the method of inactivation and the application of substrate feeding; the method of cultivation does not affect the adsorption properties of cells.

The Hsa_circ_0105558/miR-182-5p/ATF6 Cascade Affects H2O2-Triggered Oxidative Damage and Apoptosis of Human Lens Epithelial Cells.

Age-related cataract (ARC) is the prevalent cause of useful vision loss. Circular RNAs are related to ARC pathogenesis partly through their competing endogenous RNA (ceRNA) activity. Herein, we defined the action of hsa_circ_0105558 in hydrogen peroxide (H2O2)-driven apoptosis and oxidative damage in human lens epithelial SRA01/04 cells. Hsa_circ_0105558, microRNA (miR)-182-5p and activating transcription factor 6 (ATF6) were evaluated by a qRT-PCR or immunoblotting method. The hsa_circ_0105558/miR-182-5p and miR-182-5p/ATF6 relationships were predicted by bioinformatics analysis and confirmed by dual-luciferase reporter assay. Reactive oxygen species level, glutathione peroxidase level, superoxide dismutase activity, and malondialdehyde level were measured using the matched assay kits. Hsa_circ_0105558 was upregulated in human ARC lens and H2O2-exposed SRA01/04 cells. Suppression of hsa_circ_0105558 attenuated H2O2-driven SRA01/04 cell apoptosis and oxidative damage. Hsa_circ_0105558 targeted miR-182-5p, and reduced miR-182-5p expression reversed the influence of hsa_circ_0105558 depletion on H2O2-driven oxidative damage and apoptosis. ATF6 was a target of miR-182-5p, and miR-182-5p-driven downregulation of ATF6 regulated cell oxidative damage and apoptosis under H2O2 insult. Moreover, hsa_circ_0105558 functioned as a ceRNA to post-transcriptionally control ATF6 expression through miR-182-5p competition. Our study demonstrates that hsa_circ_0105558 modulates SRA01/04 cell oxidative damage and apoptosis under H2O2 insult through the miR-182-5p/ATF6 cascade.

Recognition of Immunoreactive Proteins in Leishmania infantum Amastigote-Like and Promastigote Using Sera of Visceral Leishmaniasis Patients: a Preliminary Study.

Visceral leishmaniasis (VL) is a systemic and parasitic disease that is usually fatal if left untreated. VL is endemic in different parts of Iran and is caused mainly by Leishmania infantum. This study aimed to recognition immunoreactive proteins in amastigote-like and promastigote stages of L. infantum (Iranian strain) by antibodies present in the sera of VL patients. Total protein extract from amastigote-like and promastigote cells was separated by two-dimensional electrophoresis (2DE). To detect the immunoreactive proteins, 2DE immunoblotting method was performed using different pools of VL patients' sera. Approximately 390 and 430 protein spots could be separated in 2DE profiles of L. infantum amastigote-like and promastigote stages, respectively. In immunoblotting method, approximately 295 and 135 immunoreactive proteins of amastigotes-like reacted with high antibody titer serum pool and low antibody titer serum pool, respectively. Approximately 120 and 85 immunoreactive proteins of promastigote extract were recognized using the high antibody titer sera pool and low antibody titer sera, respectively. The present study has recognized a number of antigenic diversity proteins based on the molecular weight and pH in amastigote-like and promastigote stages of L. infantum. These results provide us a new concept for further analysis development in the field of diagnosis biomarkers and vaccine targets.

COMPARISON OF THE RESULTS OF DETECTION OF ANTIBODIES TO HEPATITIS C VIRUS REAGENTS FROM DIFFERENT MANUFACTURERS

We conducted a comparative characteristic analysis of the four kits Anti-HCV Abbott (AbHCV), Anti-HCV II Roche (RoHCV), Vector-best Anti-HCV spectrum ELISA (VeHCVsp) and Vector-best screening ELISA (VeHCVsc) to select screening and confirming method measurement antibody to hepatitis C virus (anti-HCV) considering Se, Sp.We evaluated 321 samples in the three stages. At the first stage after examining 214 primary positive in screening samples we received conflicting results AbHCV, VbHCVsp and RocHCV with discrepancies in 12.6% of cases. In the second phase, we selected 67 samples with conventionally border values cut-off that is “weakly positive” or “doubtful negative” results. Differences for this group was 17.9%. In the third phase, we selected 40 conventionally border results samples (CBRS). We used for comparison immunoblotting method “Recomline HCV IgG”, Microgen diagnostic (MGHCV). We evaluated Se with positive and doubtful MGHCV results, Sp - with negative results. Se: AbbHCV = 75,0%, RocHCV = 60,0%, VbHCV = 50,0%. Sp: AbbHCV = 5,0%, RocHCV = 75,0%, VbHCV = 95,0%. To understand the causes of discrepancies of various kits, we conducted an analysis HCV antigens (Core 1, Core 2, Helicase, NS3, NS4, NS5). The uncertainty of the results for CBRS is determined by different design of recombinant molecules used each kit. Therefore, at the stage of screening samples for HCV antibodies will always be samples with conflicting results. The best decision for screening is a combination of primary screening and retesting of positive samples to use kits with the “opposite” benefits.

Current Trends in Validating Antibody Specificities for ELISpot by Western Blotting.

The enzyme-linked immunospot (ELISpot) assay is a highly useful and sensitive method to detect total immunoglobulin and antigen-specific antibody-secreting cells. In addition, this method can measure biological activity and immunological secretions from immune cells. In general, membrane-bound antigen allows binding of antibody secreted by B cells, or a membrane-bound analyte-specific antibody binds to the specific analyte (e.g., cytokines) elicited from cells added to the well containing the bound antibody. The response from added cells is then detected by using an anti-Ig antibody and a colorimetric substrate, while in the case of non-B cells, the elicited antigen is detected with appropriate antibodies and enzyme-conjugated antibodies. Specificity of antibodies binding the protein of interest is necessary to achieve correct results. Western blotting can be used for this with/without siRNA knockdown of proteins of interest or with the use of peptide inhibitors to inhibit the binding of specific antibodies to the target protein. Despite its general simplicity, western blotting is a powerful technique for immunodetection of proteins (notably low abundance proteins) as it provides simultaneous resolution of multiple immunogenic antigens within a sample for detection by specific antibodies. Now, we have plethora of immunoblotting methods to validate antibodies for ELISpot.

Neuroprotective role of vitamin B12 in streptozotocin-induced type 1 diabetic rats

Chronic hyperglycemia-induced neuropathological changes include neuronal apoptosis, astrogliosis, decrease in neurotrophic support, impaired synaptic plasticity, and impaired protein quality control (PQC) system. Vitamin B12 is indispensable for neuronal development and brain function. Several studies reported the neuroprotective effect of B12 supplementation in diabetic patients. However, the underlying molecular basis for the neuroprotective effect of B12 supplementation in diabetes needs to be thoroughly investigated. Two-month-old Sprague-Dawley rats were randomly assigned into three groups: Control (CN), diabetes (D; induced with streptozotocin; STZ), and diabetic rats supplemented with vitamin B12 (DBS; vitamin B12; 50 μg/kg) for four months. At the end of 4 months of experimentation, the brain was dissected to collect the cerebral cortex (CC). The morphology of CC was investigated with H&E and Nissl body staining. Neuronal apoptosis was determined with TUNEL assay. The components of neurotrophic support, astrogliosis, synaptic plasticity, and PQC processes were investigated by immunoblotting and immunostaining methods. H& E, Nissl body, and TUNEL staining revealed that diabetes-induced neuronal apoptosis and degeneration. However, B12 supplementation ameliorated the diabetes-induced neuronal apoptosis. Further, B12 supplementation restored the markers of neurotrophic support (BDNF, NGF, and GDNF), and synaptic plasticity (SYP, and PSD-95) in diabetic rats. Interestingly, B12 supplementation also attenuated astrogliosis, ER stress, and ameliorated autophagy-related proteins in diabetic rats. Overall, these findings suggest that B12 acts as a neuroprotective agent by inhibiting the neuropathological changes in STZ-induced type 1 diabetes. Thus, B12 supplementation could produce beneficial outcomes including neuroprotective effects in diabetic patients.

Unraveling the anti-primary dysmenorrhea mechanism of Ainsliaea fragrans Champ. extract by the integrative approach of network pharmacology and experimental verification

BackgroundThe plant Ainsliaea fragrans Champ. (A. fragrans) named "Xingxiang Tuerfeng", is a traditional herb with a long history of therapeutic practice in southern China in the treatment of gynecological diseases. PurposeThe anti-inflammatory extract of Ainsliaea fragrans Champ. (AF-ext) exhibited anti-primary dysmenorrhea (PD) activity in oxytocin-induced mice. This study aimed to unravel the underlying mechanisms of AF-ext on PD by the integrative approach of network pharmacology and experimental verification. MethodsFirst, the therapeutic targets of AF-ext are predicted using network pharmacology and molecular docking methods. Second, activity screening and immunoblotting methods were used for target validation. Then, the therapeutic effect of AF-ext on PD was evaluated using oxytocin-induced mice and uterine strips model. ResultsAF-p1, and AF-p2, the active ingredients of AF-ext, showed inhibitory effects on COX1/2 and EGFR, and all five active components showed antagonistic activity on TRPV1. AF-ext (25, 50, 100 mg/kg) could significantly reduce the number of writhing times and prolong writhing latencies in a dose-dependent manner. AF-ext inhibited spasmolytic activity in uterine strips induced by oxytocin and Ca2+ stimulation. AF-ext inhibited NF-κB/COX-2/PG pathway and activation of the NLRP3 inflammasome in PD mice. It significantly downregulated the PD-induced overexpression of p-p65/p65, p-IκBα, and COX-2 by inhibiting the NF-κB pathway. Moreover, the overexpression of NLRP3, p20/pro-Caspase 1, and p17/pro-IL-1β was greatly downregulated. ConclusionsAF-ext demonstrated anti-inflammatory, analgesic, and spasmolytic activity in the treatment of PD. It inhibited the NF-κB/COX-2/PG pathway and NLRP3 inflammasome activation in PD mice with a multi-target approach.

Pharmacological Inhibition of the AQP4 Water Channel Activity Causes an Aggravation of Alpha-Synuclein Pathology in the Substantia Nigra in a Rat Model of Parkinson’s Disease

The misfolding of the protein α-synuclein, which leads to the formation of neurototoxic oligomers and aggregates, is one of the main causes of loss of dopaminergic (DA) neurons within the substantia nigra pars compacta (SNpc) in Parkinson’s disease (PD). We previously found that pharmacological inhibition of the water channel aquaporin-4 (AQP4), participating in the mechanisms of brain clearance of amyloidogenic proteins, caused the aggravation of neurodegeneration in the nigrostriatal system and the development of motor disturbances in a lactacystin model of PD. It was hypothesized that the progression of neurodegeneration can be a result of the excessive accumulation of pathologic forms of α-synuclein due to the AQP4 inhibition. The aim of this study is to determine whether pharmacological inhibition of AQP4 activity in a rat model of preclinical PD leads to an aggravation in α-synuclein pathology. The experiments were performed on male Wistar rats. AQP4 activity was suppressed using the intracerebroventricular injection of inhibitor TGN-020. To reproduce the model of the preclinical stage of PD, a specific proteasome inhibitor lactacystin (LC) was used. It was injected bilaterally into the SNpc. Immunoblotting methods and confocal microscopy were applied. The LC model of PD was characterized by a pathologic accumulation of total water-soluble and Ser129-phosphorylated forms of α-synuclein, as well as by formation of insoluble α-synuclein aggregates in the DA-neurons of SNpc. TGN-020 caused a significant aggravation of α-synuclein pathology in the LC model of PD. It was manifested by a marked increase in the level of water-soluble and modified forms of α-synuclein and by the 1.9-fold rise in the amount of α-synuclein aggregates in SN. We suppose that the disfunction of AQP4 which is involved in glymphatic system functioning, can be one of the mechanisms leading to the neurodegeneration and accumulation of amyloidogenic proteins in brain parenchyma during PD. The water channel AQP4 might be a target for the development of new therapeutic approaches aimed at attenuation of the cytotoxicity, accumulation and distribution of α-synuclein during the development of PD-like pathology.

Pro-inflammatory GPR75 and anti-apoptotic phospholipase signaling pathways contribute to the ameliorating effect of soluble epoxide hydrolase inhibition on chronic experimental autoimmune encephalomyelitis in mice.

Soluble epoxide hydrolase (sEH) inhibition has currently emerged as a therapeutic target in the treatment of various neuroinflammatory neurodegenerative diseases, including multiple sclerosis. Previously, we reported that treatment of mice with a sEH-selective inhibitor, 1-(1-propanoylpiperidin-4-yl)-3-[4-(trifluoromethoxy)phenyl]urea; TPPU), ameliorated chronic experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein 35-55 peptide immunization followed by injection of pertussis toxin to mice via regulating pro-inflammatory and anti-inflammatory pathways in the central nervous system. This study tested the hypothesis that the pro-inflammatory G protein-coupled receptor (GPR) 75 and anti-apoptotic phospholipase C (PLC) signaling pathways also contribute to the ameliorating effect of TPPU on chronic EAE. Brains and spinal cords of phosphate-buffered saline-, dimethyl sulfoxide-, or TPPU (3 mg/kg)-treated mice were used for the measurement of sEH, GPR75, Gaq/11, activator protein (AP)-1, PLC β4, phosphoinositide 3-kinase (PI3K) p85a, Akt1, mitogen-activated protein kinase kinase (MEK) 1/2, extracellular signal-regulated kinase (ERK) 1/2, cyclic adenosine monophosphate-response element-binding protein (CREB) 1, B-cell lymphoma (Bcl)-2, semaphorin (SEMA) 3A, and myelin proteolipid protein (PLP) expression and/or activity by using the immunoblotting method. Expression of sEH, GPR75, Gaq/11, c-jun, phosphorylated c-Jun, and SEMA3A was lower, while PLCβ4, phosphorylated PI3K p85a, phosphorylated Akt1, phosphorylated MEK1/2, phosphorylated ERK1/2, phosphorylated CREB1, Bcl-2, and myelin PLP expression was higher in the tissues of TPPU (3 mg/kg)-treated mice as compared with the EAE and vehicle control groups. Inhibition of sEH by TPPU ameliorates chronic EAE through suppressing pro-inflammatory GPR75/Gaq/11/AP-1 pathway and reducing expression of the remyelination inhibitor, SEMA3A, as well as increasing anti-apoptotic PLC/PI3K/Akt1/MEK1/2/ERK1/2/CREB1/Bcl-2 pathway activity and myelin PLP expression.

Prediction of Food Sensitization in Children with Atopic Dermatitis Based on Disease Severity and Epidermal Layer Impairment

Plain Language SummaryThe study has revealed notable associations between food sensitizations (FSs) and the disease severity as well as epidermal barrier impairment in children with atopic dermatitis (AD). Specifically, the findings indicate that children with AD who were sensitized to higher numbers of food allergens (FAs) had higher SCORAD scores and greater epidermal barrier impairment. In addition, SCORAD score and stratum corneum hydration (SCH) levels at lesional skin could be predictive factors for FS in children with AD. The findings of this study suggest that clinicians should consider screening for food sensitization in children with moderate-severe AD and/or low SCH levels at lesional skins.

Apoptosis-inducing, anti-angiogenic and anti-migratory effects of a dinuclear Pd(II) complex on breast cancer: A promising novel compound

Because of the high mortality and morbidity rate of breast cancer, successful management of the disease requires synthesis of novel compounds. To this end, ongoing attempts to create new candidates include synthesis of multinuclear metal complexes. The high DNA binding affinity and cytotoxic activity of these complexes makes them promising as breast cancer treatments. This study investigated anti-growth/cytotoxic effect of the dinuclear Pd(II) complex on breast cancer cell lines (MCF-7, MDA-MB-231) using various methods of staining, flow cytometry, and immunoblotting. The study conducted colony formation, invasion, and migration assays were to assess the effect of the complex on metastasis. Increased caspase-3/7 levels and positive annexin V staining were observed in both cell lines, proving apoptosis. Altered TNFR1 and TRADD expression with caspase-8 cleavage followed by BCL-2 inactivation with loss of mitochondrial membrane potential confirmed the presence of apoptosis in MCF-7 and MDA-MB-231, regardless of p53 expression status. The results implied anti-migration properties. Finally, the study used the CAM assay to assess antiangiogenic properties and showed that the complex inhibited angiogenesis. The study concluded the dinuclear Pd(II) complex warrants further in vivo experiments to show its potential in the treatment of breast cancer.