Western Blot Results Research Articles

Serum-Derived Extracellular Vesicles for the Treatment of Severe Ocular Surface Disease

PurposeAutologous serum is widely used for the treatment of severe ocular surface disease with mixed efficacy. Extracellular vesicles (EVs) are small membrane bound structures present in all body fluids, including serum. This study compared the proteomic, metabolomic, and inflammatory cytokine composition of serum-derived EVs (SDEVs) to that of the soluble free protein fraction and the subsequent capacity of SDEVs to induce corneal epithelial cell migration and inflammation. MethodsSDEVs were isolated from human serum using size exclusion chromatography. SDEVs were analyzed using nanoparticle tracking analysis, transmission electron microscopy, and western blotting. The effects of SDEVs on corneal epithelial cell migration were tested using a standard scratch assay. Inflammatory cytokines in SDEVs and the free protein fraction were quantified using a microarray. A mutli-omics approach was further used to define SDEV cargo. The ability of SDEVs to modulate inflammation in corneal epithelial cells was quantified using ELISAs. ResultsWestern blot and TEM confirmed the presence of SDEVs. Proinflammatory cytokines, along with complement proteins and TGF-β, were decreased in SDEVs compared to serum. Metabolites present in SDEVs were mostly involved in amino acid biosynthesis, the TCA cycle and oxidative phosphorylation. SDEVs exhibited pro-migratory effects similar to serum however, SDEVs did not induce secretion of IL-6 or IL-8. ConclusionsSDEVs exhibit reduced levels of pro-inflammatory cytokines while retaining the beneficial wound healing properties of serum. Unlike serum, SDEVs do not induce inflammation. SDEVs may represent an alternative option for patients with severe ocular surface disease where traditional autologous serum has failed.

Topical hADSCs-HA Gel Promotes Skin Regeneration and Angiogenesis in Pressure Ulcers by Paracrine Activating PPARβ/δ Pathway.

Pressure ulcer is common in the bedridden elderly with high mortality and lack of effective treatment. In this study, human-adipose-derived-stem-cells-hyaluronic acid gel (hADSCs-HA gel) was developed and applied topically to treat pressure ulcers, of which efficacy and paracrine mechanisms were investigated through in vivo and in vitro experiments. Pressure ulcers were established on the backs of C57BL/6 mice and treated topically with hADSCs-HA gel, hADSCs, hyaluronic acid, and normal saline respectively. The rate of wound closure was observed continuously during the following 14 days and the wound samples were obtained for Western blot, histopathology, immunohistochemistry, and proteomic analysis. Human dermal fibroblasts (HDFs) and human venous endothelial cells (HUVECs) under normal or hypoxic conditions were treated with conditioned medium of human ADSCs (ADSC-CM), then CCK-8, scratch test, tube formation, and Western blot were conducted to evaluate the paracrine effects of hADSCs and to explore the underlying mechanism. The in vivo data demonstrated that hADSCs-HA gel significantly accelerated the healing of pressure ulcers by enhancing collagen expression, angiogenesis, and skin proliferation. The in vitro data revealed that hADSCs strengthened the proliferation and wound healing capabilities of HDFs and HUVECs, meanwhile promoted collagen secretion and tube formation through paracrine mode. ADSC-CM was also proved to exert protective effects on hypoxic HDFs and HUVECs. Besides, the results of proteomic analysis and Western blot elucidated that lipid metabolism and PPARβ/δ pathway mediated the healing effect of hADSCs-HA gel on pressure ulcers. Our research showed that topical application of hADSCs-HA gel played an important role in dermal regeneration and angiogenesis. Therefore, hADSCs-HA gel exhibited the potential as a novel stem-cell-based therapeutic strategy of treating pressure ulcers in clinical practices.

Anti-Radiofibrosis Effect of Dicliptera chinensis Polysaccharide on Rat Dermal Fibroblasts Via The TGF-β1/Smads/CTGF Signaling Pathway

Radiotherapy is an effective treatment for head and neck cancer; however, irradiated normal tissues are inevitably damaged, resulting in skin radioactive fibrosis. Dicliptera chinensis polysaccharide (DCP), the primary active compound extracted from the natural medicinal Dicliptera chinensis, exhibits antioxidant, anti-inflammatory, and anti-radiation properties. In this study, we investigated the protective effects of DCP against radioactive fibrosis in rat dermal fibroblasts (RDF) and explored the underlying mechanisms involved. RDFs were treated with DCP, and the CCK8 assay was used to determine cellular activity. The rates of apoptosis and cell cycle progression were detected using flow cytometry. mRNA expression levels were quantified using real-time polymerase chain reaction. Protein levels were analysed through Western blotting and immunofluorescence staining RESULTS: DCP reduced radiation-induced apoptosis, and the cell cycle G2/M arrest was alleviated. Furthermore, DCP decreased the expression of key fibrosis-related markers, including α-SMA, TGF-β1, Smad3, and CTGF. DCP exhibits a protective effect against radiation-induced fibrosis.

Tile: Construction of a specific nanoprobe for scavenging ROS in hypobaric hypoxia induced brain injury of mice

The prevention and treatment of hypobaric hypoxia brain injury (HHBI) remains an unprecedented challenge due to the complex oxidative stress response at the damage site. In this study, RuCO phthalocyanine compound (RuPc) and bovine serum albumin (BSA) were self-assembled to obtain RuPc-BSA nanoparticles for HHBI therapy. As a nanoprobe carrying and storing carbon monoxide (CO), RuPc-BSA delivers CO to pathologically damaged areas of the brain. CO specifically attaches itself to the heme functional groups on mitochondria and restricts the source of reactive oxygen species (ROS) generation. RuPc-BSA nanoparticles have been demonstrated in vitro to exhibit amazing stability as well as remarkable scavenging activity on hydroxyl radical, superoxide anion, and hydrogen peroxide. In vivo experiments showed that ROS levels in the brain of HHBI rats pretreated with RuPc-BSA decreased significantly, and neuronal function and oxidative stress levels were alleviated. Western blot and qRT-RCR results indicated that RuPc-BSA restricted the protein levels of Keap1, whereas enhanced the gene and protein levels of Nrf2. This study demonstrated that RuPc-BSA can ameliorate HHBI of mice by scavenging ROS partly via activating Keap1/Nrf2 signaling pathway.

Herbal medicine Ninjinyoeito inhibits RANKL-induced osteoclast differentiation and bone resorption activity by regulating NF-κB and MAPK pathway

ObjectivesOsteoporosis is a systemic bone metabolism disorder characterized by decreased bone mass and strength. Osteoclasts (OCs) are giant multinucleated cells that regulate bone homeostasis by degrading bone matrix. Excessive OC differentiation and activity can lead to serious bone metabolic disorders including osteoporosis. Current treatments, including antiresorptive drugs, exert considerable adverse effects, including jaw osteonecrosis. Herbal medicines, such as Ninjinyoeito (NYT), may also offer efficacy, but with fewer adverse effects. In this study, we investigated NYT's effects on osteoclastogenesis. MethodsTartrate-resistant acid phosphatase (TRAP) staining and bone resorption assays were performed to examine NYT's effects on OC differentiation and function. OC-related gene expression at mRNA and protein levels was investigated to confirm NYT's inhibitory action against osteoclastogenesis. We also demonstrated involvement of signaling pathways mediated by IκBα and mitogen-activated protein kinases (MAPK) [extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38] and showed nuclear translocation of nuclear factor of activated T-cell cytoplasmic 1 (NFATc1) and nuclear factor kappa B (NF-κB) p65 during osteoclastogenesis. ResultsTRAP staining and bone resorption assays confirmed that NYT significantly inhibited OC differentiation and function. Western blot and RT-PCR results showed that NYT ameliorated osteoclastogenesis by suppressing mRNA and protein level expression of OC-related genes. Moreover, blots and immunocytochemistry (ICC) data clarified that NYT abrogates signaling pathways mediated by IκBα and MAPK (ERK, JNK, p38), and demonstrated nuclear translocation of NFATc1 and NF-κB p65 during OC differentiation. ConclusionsThese findings suggest NYT is an alternative therapeutic candidate for treating osteoporosis.

Effects of ATG5 and ATG7 Knockout on Ferroptosis Sensitivity of RPMI-8226 Cells

To investigate the effects of ATG5 and ATG7 genes on the sensitivity of multiple myeloma (MM) cell line RPMI-8226 cells to ferroptosis. CRISPR/Cas9 technology was used to knock out the autophagy key genes ATG5 and ATG7 in RPMI-8226 cells. Western blot was used to identify gene knockout cells, and detect the expression changes of autophagy-related proteins P62 and LC3B. Flow cytometry was used to detect the change of sensitivity of gene knockout cells to RSL3. The content of intracellular ferrous ions and reactive oxygen species (ROS) level in gene knockout cells were detected. Western blot result confirmed that ATG5 and ATG7 genes were knocked out successfully in RPMI-8226 cells. The result of flow cytometry showed that the cell viability of RPMI-8226 cells was dose-dependent on different concentrations of RSL3 (r =-0.969). RSL3 (10 μmol/L) was used to induce ferroptosis in cells of control group and gene knockout groups, then the cell viability in gene knockout groups were both higher than control group after 48 hours (both P < 0.001). After knocking out the ATG5 and ATG7 genes, the content of intracellular Fe2+ decreased significantly compared with control group (both P < 0.01), and the ROS level also decreased (both P < 0.001). Knockout of ATG5 and ATG7 genes can inhibit the ferroptosis of MM cells, and LAP pathway may be involved in the regulation.

Integrated multi-omics approach and pathological analyses provide new insights into the hepatic injury and metabolic alterations in Saanen goats after dietary exposure to aflatoxin B1

Exploring the toxicity and metabolic mechanisms of Aflatoxin B1 (AFB1) in ruminants can help to develop strategies to prevent or reduce the transfer of the toxin and its metabolites to milk and meat. This study aimed to explore the effects of 3 concentrations of dietary AFB1 (0, 50, and 500 μg/kg) on hepatic injury and metabolism in Saanen goats via histological examination, Western blot analysis, as well as integrated multi-omics techniques. Eighteen Saanen goats were assigned to 1 of 3 treatments and the AFB1 challenge lasted for 14 d. Results showed that the liver tissue was enlarged and the relative organ index of the liver was linearly increased with elevated AFB1 levels. The hepatocyte apoptosis rate was significantly increased after AFB1 exposure and the Western blotting results revealed that both the external apoptotic pathway and mitochondrial-mediated intrinsic apoptotic pathway might be involved in AFB1-induced hepatocyte apoptosis. There were 251, 269, 154 significant differentially expressed genes (DEGs) and 340, 596, 127 significant differential metabolites for the control (C; 0 μg/kg) and low-dose (L; 50 μg/kg), C and high-dose (H; 500 μg/kg), L and H comparisons. The DEGs annotated were mainly involved in the cell part, cell, single-organism process, cellular process, binding, and other functional categories. The identified metabolites primarily belonged to glycerophospholipids, prenol lipids, carboxylic acids and derivatives. Integrative analysis of transcriptomics and metabolomics revealed that glycerophospholipids metabolism and choline metabolism in cancer were the most affected pathways related to AFB1 exposure. The identified differential metabolites, DEGs, and pathways might have played a crucial role in the hepatic injury induced by AFB1 in goats.

Xiaobugan decoction prevents CCl4-induced acute liver injury by modulating gut microbiota and hepatic metabolism

BackgroundThe liver plays a crucial role in detoxification and metabolism. When its capacity to metabolize foreign substances is exceeded, it can lead to acute liver injury (ALI). Therefore, preventing liver disease and maintaining daily liver health are of utmost importance. Xiaobugan Decoction (XBGD), a traditional Chinese medicine (TCM) formula, is recorded in ‘Fuxingjue’, is used in folk practice to promote liver health and regulate respiration. However, the hepatoprotective mechanisms of XBGD remained unclear. PurposeWe investigated the prophylactic and hepatoprotective effects of XBGD and explored its related molecular mechanisms using a mouse model of carbon tetrachloride (CCl4)-induced ALI. Study design and methodsXBGD composition was determined using analytical methods, and the main compounds were identified using ultra-high-performance liquid chromatography coupled with Q-Exactive focus mass spectrum (UHPLC-QE-MS) and high-performance liquid chromatography (HPLC). A CCl4-induced L02 cell injury model was employed to explore the protective effects of XBGD on liver cells, and a CCl4-induced ALI mouse model was used to investigate the hepatoprotective effects of XBGD. ResultsCellular experiments demonstrated that XBGD had a protective function against L02 cell damage by increasing cell viability, restoring alanine aminotransferase (ALT), aspartate aminotransferase (AST), and superoxide dismutase (SOD) levels, reducing malondialdehyde (MDA) content, and improving mitochondrial membrane potential (ΔΨm). In the mouse ALI model, XBGD prevented ALI by reducing ALT, AST, and alkaline phosphatase (ALP) levels and inhibiting oxidative stress. Quantitative real-time polymerase chain reaction (qPCR), immumohistochemical staining and western blotting results revealed that XBGD exerted hepatoprotective effects by reducing inflammatory responses and inhibiting cell apoptosis. Furthermore, 1H-NMR metabolomics indicated that XBGD regulates hepatic and intestinal metabolism, whereas 16S rDNA sequencing demonstrated the regulatory effects of XBGD on the gut microbiota. Correlation analysis highlighted the close relationship among gut microbiota, metabolites, and ALI indicators. ConclusionsXBGD is a promising TCM for the prevention of CCl4-induced ALI via regulation of microbiota and metabolism. This study provides a new perspective on the development of hepatoprotective measures and the prevention of liver disease in daily life.

Comparative Analysis of Intestinal TLR4 Gene Expression and Functional Verification in Lepus yarkandensis and Oryctolagus cuniculus.

Lepus yarkandensis live year-round in harsh desert environments and are less susceptible to enteritis. The living conditions of Oryctolagus cuniculus in captivity were suitable, but they were highly susceptible to death by Gram-negative bacteria infected with inflammatory bowel disease complex.TLR4 is closely related to the occurrence of enteritis, and the neighbor-joining topology based on the 12S rDNA sequences showed that the relationship between O. cuniculus and L. yarkandensis is as high as 98%.Therefore, we chose O. cuniculus and L. yarkandensis for comparative study.The purpose of this study was to investigate the role of Toll-like receptor 4 (TLR4) in the regulation of immunity and inflammation in the intestinal tract of L. yarkandensis. In this study, the TLR4 gene was cloned for the first time in the colon of L. yarkandensis. The expression of TLR4 in the intestinal tissues of L. yarkandensis and O. cuniculus was detected by histological observation, real-time fluorescence quantification PCR(qRT-PCR), and protein blotting (Western blot).An LPS-induced cell inflammation model was constructed in vitro, and ELISA was used to examine the effect of pEGFP-N1-TLR4 and siRNA knockout on the anti-inflammatory ability of the TLR4 gene. The results showed that the open reading frame of the L. yarkandensis TLR4 gene was 2520bp in length. Compared with the sequence of O. cuniculus, there were 15 differences in the TLR4 amino acid sequence of L. yarkandensis, 12 of which occurred in the LRR domain and 2 in the TIR domain, and the sequence changed from G to D at position 298. Immunohistochemistry showed that TLR4 was mainly expressed in the epithelial cells of the colon L. yarkandensis, and the expression level of TLR4 in the cecum and colon was significantly lower compared with that of O. cuniculus. qRT-PCR and Western blot results showed that the expression level of TLR4 in the colon of L. yarkandensis was significantly lower than that of O. cuniculus. At the cellular level, ELISA showed that overexpression of the TLR4 protein in L. yarkandensis could reduce the LPS-induced inflammatory response. Therefore, according to the above results, the protein structure and function of L. yarkandensis TLR4 may be different due to the change of nucleotide, which affects its binding with LPS and the activation of downstream molecules, so that L. yarkandensis is not prone to enteritis and can adapt to the harsh desert environment for a long time. This study also laid the foundation for improving the disease resistance of O. cuniculus and promoting the development and utilization of genes in L. yarkandensis.

IDH1/MDH1 deacetylation promotes NETosis by regulating OPA1 and autophagy

BackgroundAs a heterogeneous and life-threatening disease, the pathogenesis of acute liver failure (ALF) is complex. Our previous study has shown that IDH1/MDH1 deacetylation promotes ALF by regulating NETosis (a novel mode of cell death). In this article, we explore the manners of IDH1/MDH1 deacetylation regulates NETosis. MethodsIn vitro experiments, the formation of NETs was detected by immunofluorescence staining and Western blotting. LC3 fluorescence staining was used to detect autophagosome formation. To observe mitochondrial morphology, cells were stained by Mito-Tracker Red. Western blotting was used to detect the levels of autophagy protein and mitochondrial dynamin. In vivo experiments, the ALF model in mouse was established with LPS/D-gal, and the formation of NETs was detected by immunofluorescence staining and Western blotting. The autophagy levels were detected by Western blotting in liver samples. ResultsIn dHL-60 cells, Western blotting results showed that the expression of OPA1 was higher in the IDH1/MDH1 deacetylated group compared with the IDH1/MDH1 WT group. And histone deacetylase inhibitor 6 (HDAC6i, ACY1215) decreased the expression level of OPA1 in IDH1/MDH1 deacetylated group. IDH1/MDH1 deacetylation increased the expression levels of both LC3B-II and Beclin 1, while decreasing the expression level of P62. It was reversed by ACY1215. Combined with our previous experiments, IDH1/MDH1 deacetylation upregulated autophagy concomitant with the increased expression of the markers of NETs formation. In a mouse model of ALF, ACY1215 further decreased the expression levels of LC3B-II and Beclin 1, while increasing the expression level of P62 in IDH1/MDH1 deacetylated mice. ConclusionsIDH1/MDH1 deacetylation promoted NETosis by regulating autophagy and OPA1 in vitro. The regulation of neutrophil autophagy on NETosis during IDH1/MDH1 deacetylation might be masked in mice. ACY1215 might attenuate NETosis by regulating neutrophil autophagy, which alleviated ALF aggravated by IDH1/MDH1 deacetylation.

Bioinformatics analysis of ferroptosis in frozen shoulder

ObjectivesFrozen shoulder is a common shoulder disease that significantly affects the patient’s life and work. Ferroptosis is a new type of programmed cell death, which is involved in many diseases. However, there have been no studies reporting the relationship between frozen shoulders and ferroptosis. This study identified potential molecular markers of ferroptosis in frozen shoulders to provide more effective strategies for the treatment of frozen shoulders.MethodsGSE238053 was downloaded from the Gene Expression Omnibus (GEO) dataset and intersected with ferroptosis genes to obtain differentially expressed genes (DEGs). The signaling pathways and biological functions of DEGs were performed by WebGestalt and Metascape. The interactions related to these DEGs and the key genes between frozen shoulders and ferroptosis was performed by STRING and Cytoscape. A frozen shoulders rat model was used to validate our predicted genes, Western Blot and qRT-PCR was used to assess the expression levels of our genes of interest.ResultsA total of 34 DEGs between GSE238053 and Ferroptosis Database were obtained, most of which were involved in the HIF-1 signaling pathway and inflammatory response. A protein–protein interaction network was obtained by Cytoscape and the key genes (IL-6, HMOX1 and TLR4) were screened by MCODE. Our results of Western Blot showed that the protein expression level of TLR4 and HMOX1 were elevated, and the protein level of IL-6 decreased in frozen shoulders rat model. The mRNA level after frozen shoulders showed that IL-6 was upregulated, whereas TLR4 and HMOX1were downregulated.ConclusionsThe results demonstrated that ferroptosis may affect the pathological process of frozen shoulders through these signaling pathways and genes. The identification of IL-6, HMOX1 and TLR4 genes can provide new therapeutic targets for frozen shoulders.

Isovaleramide attenuates ethylene glycol poisoning-induced acute kidney injury and reduces mortality by inhibiting alcohol dehydrogenase activity in rats.

We explored the potential value of the alcohol dehydrogenase (ADH) inhibitor isovaleramide (ISO) in the treatment of acute ethylene glycol (EG) poisoning-induced acute kidney injury. Sprague-Dawley rats were divided into the control, EG, EG + ISO (10mg/kg) and EG + ISO (20 mg/kg) groups. It is found that ISO intervention significantly reduced the ADH activity in liver tissue by using visible spectrophotometry, inhibited the in vivo metabolism of EG by using gas chromatography, lowered the levels of toxic metabolites glycolic acid and oxalic acid by using high-performance liquid chromatography and decreased the expression of kidney injury markers serum creatinine (sCr), KIM-1, neutrophil gelatinase-associated lipocalin (NGAL) and liver fatty acid-binding protein (L-FABP) by ELISA. Additionally, Western blotting results showed that ISO down-regulated the expression of apoptotic factors Bax and cleaved caspase-3 in the kidneys and upregulated the expression of antiapoptotic factor Bcl-2. Pizzolato staining and polarized light microscopy results revealed the reduced deposition of calcium oxalate crystals in the kidney tubules. Using haematoxylin and eosin (H&E), periodic acid-Schiff (PAS) and Masson staining, we found attenuated kidney tissue pathological injury. Finally, ISO significantly reduced the mortality rate. In conclusion, ISO has the potential to be a valuable drug for the treatment of EG poisoning-induced acute kidney injury.

Dihydrotestosterone induces reactive oxygen species accumulation and mitochondrial fission leading to apoptosis of granulosa cells

Dihydrotestosterone (DHT), which has significant androgenic activity，is a major player in follicle development and ovary function in females. However, an excess of androgens may result in increased follicular apoptosis with adverse effects on female fertility. This study aimed to explore the mechanism by which DHT induces apoptosis in human ovarian granulosa cells (GCs). The association between DHT and GC apoptosis was explored by the construction of rat models of polycystic ovary syndrome (PCOS). It was found that serum DHT levels were negatively correlated with thickness of the GC layer in PCOS model rats (R2=0.8342, p＜0.0001), compared with control rats, together with significant increases in cofactors (Fis1: p=0.008; MFF: p=0.044). The GC SVOG cell line was used to clarify the mechanism by which DHT influenced GC apoptosis in in vitro experiments. The results confirmed that apoptosis in SVOG cells was positively associated with the DHT dose. The expression of the autophagy-related proteins LC3A/B (p=0.027) and the proapoptotic protein Bax (p=0.0095) were increased, while that of the anti-apoptotic protein Bcl-2 (p=0.0005) was decreased in the high-dose DHT group. ROS levels were significantly increased (p=0.0237) and the mitochondrial membrane potential ΔΨm was decreased (p=0.0194). Moreover, ultrastructural analysis of the mitochondria indicated significant damage. The results of RT-qPCR and western blotting showed that two fission cofactor-Fis1（p=0.034） and MFF (p=0.039) were significantly increased after treatment with high doses of DHT. Even though the overall expression of Drp1 did not change significantly (p=0.5961), that of activated Phosphor-Drp1(Ser616) was significantly increased (p=0.046), while the expression of Phosphor-Drp1 (Ser637) was markedly reduced (p=0.007) following exposure to high concentrations of DHT. All these effects could be reversed by the Drp1 inhibitor Mdivi-1. These findings indicated the impact of DHT on ROS aggregation and mitochondrial fission, resulting in GC apoptosis. An imbalance in Drp1 phosphorylation may be the key link in DHT-induced excessive mitochondrial fission.

Supercritical CO2 extracts of propolis inhibits tumor proliferation and Enhances the immunomodulatory activity via activating the TLR4-MAPK/NF-κB signaling pathway

Propolis is a natural immunomodulator with anticancer activity. This study investigated the immunomodulatory mechanism and anti-tumor activity of supercritical CO2 extracts of propolis (SEP) in tumor-bearing immunosuppression mice. We used cyclophosphamide (CTX) to construct the immunosuppressive mice model and then inoculated them with CT26 cells to build the CT26 tumor-bearing immunosuppression mice model. Upon treatment with SEP, tumor proliferation in mice was markedly suppressed, with tumor volumes decreasing from 1881.43 mm3 to 1049.95 mm3 and weights reducing from 2.07 g to 1.13 g. Concurrently, the immune system recovered well, and the spleen and thymus indexes increased significantly. The total T lymphocyte (CD3+ T cell) contents in the spleen and blood recovered from 11.88 % to 21.19 % and 15.32 % to 22.19 %, respectively. In addition, the CD4+ /CD8+ ratio has returned to a healthy level, 3.12 in the spleen and 5.42 in the blood. The levels of IL-1β, IL-6, and TNF-α were increased by 2.17, 2.76, and 7.15 times in the spleen, 2.76, 1.92, and 3.02 times in the serum. Moreover, the western blot results showed that SEP treatment increased the expression of toll-like receptor 4 (TLR4) and the phosphorylation of p38, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p65. These results indicated that SEP activated the immune activity of RAW 264.7 macrophages through the TLR4-mitogen-activated protein kinase (MAPK)/nuclear factor kappa B (NF-κB) signaling pathway to exert immunomodulatory function and inhibit tumor proliferation. This study facilitated the further application of SEP as a potential immunomodulatory and anti-tumor functional food.

Cloning and Expression Analysis of TGF-β Type I Receptor Gene in Hyriopsis cumingii.

The TGF-β signaling pathway plays an important role in wound healing and immune response. In this study, a TGF-β type I receptor (TGF-βRI) homolog was cloned and characterized from freshwater mussel Hyriopsis cumingii. The full-length cDNA of the TGF-β RI gene was 2017 bp, with a 1554 bp open reading frame (ORF), and encoded 517 amino acids. The predictive analysis further identified distinct regions within the TGF-βRI protein: a signal peptide, a membrane outer region, a transmembrane region, and an intracellular region. Real-time quantitative PCR results showed that the TGF-β RI gene was expressed in all tissues of healthy mussels. The transcripts of TGF-β RI in hemocytes and hepatopancreas were significantly up-regulated at different periods after stimulation with Aeromonas hydrophila and peptidoglycan (PGN) (P < 0.05). The mRNA expression of TGF-β RI progressively increased from day 1 to day 10 after trauma (P < 0.05), and it returned to the initial level by day 15. The expression levels of TGF-β , Smad5, MMP1/19, and TIMP1/2, but not Smad3/4, were significantly up-regulated at different time points after trauma. However, the expression levels of TGF-β , MMP1/19, and TIMP2 were decreased after treatment with the inhibitor SB431542. Furthermore, the recombinant TGF-βRI proteins were expressed in vitro and existed in the form of inclusion bodies. Western blotting results showed that TGF-βRI proteins were expressed constitutively in various tissues of mussels, and their expression was up-regulated after trauma, which was consistent with the mRNA expression trend. These results indicate that TGF-β RI is involved in the process of wound repair and immune response.

Effects of normal mitochondrial transplantation on proliferation, apoptosis and stemness of triple-negative breast cancer cells

Objectives: To observe the mitochondrial morphology of normal and triple-negative breast cancer cells, extract mitochondria from normal cells, and investigate the effects of mitochondrial transplantation on proliferation, apoptosis, and stemness of triple-negative breast cancer cells. Methods: The morphology of mitochondria was observed by transmission electron microscope. Mitochondria were extracted by mitochondrial extraction kit, mitochondrial protein was identified by western blot, and mitochondrial activity was detected by mitochondrial membrane potential detection kit. MitoTracker Green or MitoTracker Deep Red fluorescent probes were used to label the mitochondria of living cells, and the degree of mitochondria entering LTT cells was observed by confocal laser microscopy at 12, 24, and 96 hours. The effects of mitochondrial transplantation on proliferation, apoptosis, and stemness of breast cancer cells were examined by CCK8, colony formation assay, flow cytometry, and sphere formation assay after 24 hours of mitochondrial transplantation. Results: The mitochondria of normal cells were rod-shaped or elongated, while the mitochondria of triple-negative breast cancer cells were swollen and vacuolated. Western blot results showed that cytochrome c oxidase subunit I (MT-CO1) protein encoded by mitochondria was present in the isolated mitochondria. The content of heat shock protein 60 (HSP60) was higher in mitochondria than that in cytoplasm. The result of the multi-mode microplate reader showed that the content of mitochondrial J-aggregates/monomer was 1.67±0.06, which was significantly higher than 0.35±0.04 of the control group (P＜0.001). Exogenous mitochondria were observed in LTT cells at 12, 24, and 96 hours after mitochondrial transplantation. The results of the CCK8 experiment showed that OD450 of LTT cells was 0.27±0.13 after 48 hours transplantation, which was lower than 0.62±0.36 of the control group (P=0.023). The OD450 of MDA-MB-468 cells was 0.30±0.03, which was lower than 0.65±0.10 of the control group (P=0.004). After 120 hours of mitochondrial transplantation, OD450 in both groups was still significantly lower than that in the control group (P＜0.01). The number of clones formed by mitochondrial transplantation of LTT cells was 21.33±7.31, which was lower than 35.22±13.59 of the control group (P=0.016). Flow cytometry showed that the early apoptosis rate of LTT cells was (30.07±2.15)% after 24 hours of mitochondrial transplantation, which was higher than 2.07±1.58 of the control group (P＜0.001). The proportion of early apoptosis in MDA-MB-468 cells was 24.47%±5.22%, which was higher than (7.83±2.06)% in the control group (P=0.007). In addition, the number of mitochondria transplanted LTT cells into the cell sphere was 46.25±5.40, which was significantly lower than 62.58±6.43 of the control group (P＜0.001). Conclusion: Normal mitochondria can enter triple-negative breast cancer cells by co-culture, inhibit the proliferation and stemness of triple-negative breast cancer cells, and promote the apoptosis of triple-negative breast cancer cells.

GPR40 agonist ameliorates neurodegeneration and motor impairment by regulating NLRP3 inflammasome in Parkinson’s disease animal models

Parkinson's disease (PD) is characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra (SN) and accumulation of intracellular α-synuclein (ɑ-syn) aggregates known as Lewy bodies and Lewy neurites. Levels of polyunsaturated fatty acids (PUFAs) have previously been shown to be reduced in the SN of PD patients. G protein-coupled receptor 40 (GPR40) serves as a receptor for PUFAs, playing a role in neurodevelopment and neurogenesis. Additionally, GPR40 has been implicated in several neuropathological conditions, such as apoptosis and inflammation, suggesting its potential as a therapeutic target in PD.In this study, we investigated the neuroprotective effects of the GPR40 agonist, TUG469 in PD models. Our results demonstrated that TUG469 reduces the neurotoxicity induced by 6-OHDA in SH-SY5Y cells. In 6-OHDA-induced PD model mice, TUG469 treatment improved motor impairment, preserved dopaminergic fibers and cell bodies in the striatum (ST) or SN, and attenuated 6-OHDA-induced microgliosis and astrogliosis in the brain. Furthermore, in a PD model involving the injection of mouse ɑ-syn fibrils into the brain (mPFFs-PD model), TUG469 treatment reduced the levels of pSer129 ɑ-syn, and decreased microgliosis and astrogliosis. Our investigation also revealed that TUG469 modulates inflammasome activation, apoptosis, and autophagy in the 6-OHDA-PD model, as evidenced by the results of RNA-seq and western blotting analyses.In summary, our findings highlight the neuroprotective effects of GPR40 agonists on dopaminergic neurons and their potential as therapeutic agents for PD. These results underscore the importance of targeting GPR40 in PD treatment, particularly in mitigating neuroinflammation and preserving neuronal integrity.

WDR64, a testis-specific protein, is involved in the manchette and flagellum formation by interacting with ODF1

The WD40 repeat (WDR) domain is present in a wide range of proteins, providing sites for protein‒protein interactions. Recent studies have shown that WDR proteins play indispensable roles in spermatogenesis, such as in spermatocyte division, sperm head formation and flagellar assembly. In this study, we identified a novel testis-specific gene, WDR64, which has the typical characteristics of WD40 proteins with two β-propellers, and is highly conserved in Mammalia. RT-PCR and Western blot results revealed that WDR64 was highly expressed in testis. WDR64 protein was weakly expressed at postnatal Day 7, increased substantially at postnatal Day 28 and maintained at high levels thereafter. Further immunofluorescence demonstrated that WDR64 was localized posterior to the nucleus in steps 8–14 spermatids in line with the dynamic localization of manchette, moved to the flagella in steps 15–16 spermatids, and localized at the midpiece of the flagellum in mature spermatozoa. To explore the function of WDR64, we performed immunoprecipitation‒mass spectrometry (IP‒MS) to screen its interacting proteins and found that WDR64 interacted with ODF1 to form a complex. The WDR64/ODF1 complex is located at the manchette during nucleus shaping and finally at the midpiece of the mature spermatozoa tail, suggesting that it may be involved in the assembly of the manchette and flagella during spermiogenesis. Our findings provide the first understanding of the expression pattern of WDR64 and its potential molecular mechanism in spermiogenesis.

α-Cyperone Antagonizes Intestinal Mucosal Inflammatory Response Through Modulation of TLR4/NF-κB Signaling Pathway to Alleviate Crohn's Disease-Like Colitis in Mice

To investigate the effect and potential mechanisms of α-cyperone (CYP) on Crohn's disease (CD) -like colitis induced by 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) in mice. The mice were randomly and evenly divided into wild type (WT), TNBS, CYP and 5-aminosalicylic acid (5-ASA) groups, with 10 mice in each group. The symptoms of enteritis, the function and structure of the intestinal barrier, and the expression levels of inflammatory factors, including interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and gamma-interferon (IFN-γ), in the colon were assessed. The lipopolysaccharide (LPS)-induced inflammation model of Caco2 cells was constructed and the cells were divided into Control, LPS and LPS+CYP groups. The expression levels of tight junction protein and inflammatory factors in each group were assessed. Gene Ontology (GO) functional enrichment analysis was conducted to predict the possible pathways of action and potential molecular mechanisms of CYP, and to verify them in vivo and in vitro. In the in vivo study, compared with those of the TNBS group, the body mass and colon length of mice in the CYP group and the 5-ASA group were significantly increased, while the disease activity scores and histological inflammation scores were significantly decreased (P<0.05). The level of lucifcein-glucan isothiocyanate and the bacterial translocation rate (in the liver, the spleen, and mesenteric lymph nodes) were significantly decreased, while the transepithelial electric resistance (TEER) value and the expression levels of zonula occluden protein-1 (ZO-1), and claudin-1 were significantly increased (P<0.05). The expression of inflammatory factors was significantly decreased (P<0.05). In the in vitro study, compared with those of the LPS group, the TEER value and the expression of ZO-1 and claudin-1 in the Caco2 cells in the LPS+CYP group were significantly increased (P<0.05). The expression of inflammatory factors was significantly decreased (P<0.05). Enrichment analysis showed that CYP was correlated with inflammatory response (P<0.001). Western blot results showed that CYP could significantly reduce the expression of key proteins in toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signaling pathway in vivo and in vitro (P<0.05). CYP may protect the intestinal barrier by antagonizing the inflammatory response of the intestinal mucosa through regulating the expression of the TLR4/NF-κB signaling pathway, thereby alleviating TNBS-induced CD-like colitis in mice.

Glucocorticoids Rapidly Modulate CaV1.2-Mediated Calcium Signals through Kv2.1 Channel Clusters in Hippocampal Neurons.

The precise regulation of Ca2+ signals plays a crucial role in the physiological functions of neurons. Here, we investigated the rapid effect of glucocorticoids on Ca2+ signals in cultured hippocampal neurons from both female and male rats. In cultured hippocampal neurons, glucocorticoids inhibited the spontaneous somatic Ca2+ spikes generated by Kv2.1-organized Ca2+ microdomains. Furthermore, glucocorticoids rapidly reduced the cell surface expressions of Kv2.1 and CaV1.2 channels in hippocampal neurons. In HEK293 cells transfected with Kv2.1 alone, glucocorticoids significantly reduced the surface expression of Kv2.1 with little effect on K+ currents. In HEK293 cells transfected with CaV1.2 alone, glucocorticoids inhibited CaV1.2 currents but had no effect on the cell surface expression of CaV1.2. Notably, in the presence of wild-type Kv2.1, glucocorticoids caused a decrease in the surface expression of CaV1.2 channels in HEK293 cells. However, this effect was not observed in the presence of nonclustering Kv2.1S586A mutant channels. Live-cell imaging showed that glucocorticoids rapidly decreased Kv2.1 clusters on the plasma membrane. Correspondingly, Western blot results indicated a significant increase in the cytoplasmic level of Kv2.1, suggesting the endocytosis of Kv2.1 clusters. Glucocorticoids rapidly decreased the intracellular cAMP concentration and the phosphorylation level of PKA in hippocampal neurons. The PKA inhibitor H89 mimicked the effect of glucocorticoids on Kv2.1, while the PKA agonist forskolin abrogated the effect. In conclusion, glucocorticoids rapidly suppress CaV1.2-mediated Ca2+ signals in hippocampal neurons by promoting the endocytosis of Kv2.1 channel clusters through reducing PKA activity.