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.

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.

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.

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.

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.

A tick saliva serpin, IxsS17 inhibits host innate immune system proteases and enhances host colonization by Lyme disease agent.

Lyme disease (LD) caused by Borrelia burgdorferi is among the most important human vector borne diseases for which there is no effective prevention method. Identification of tick saliva transmission factors of the LD agent is needed before the highly advocated tick antigen-based vaccine could be developed. We previously reported the highly conserved Ixodes scapularis (Ixs) tick saliva serpin (S) 17 (IxsS17) was highly secreted by B. burgdorferi infected nymphs. Here, we show that IxsS17 promote tick feeding and enhances B. burgdorferi colonization of the host. We show that IxsS17 is not part of a redundant system, and its functional domain reactive center loop (RCL) is 100% conserved in all tick species. Yeast expressed recombinant (r) IxsS17 inhibits effector proteases of inflammation, blood clotting, and complement innate immune systems. Interestingly, differential precipitation analysis revealed novel functional insights that IxsS17 interacts with both effector proteases and regulatory protease inhibitors. For instance, rIxsS17 interacted with blood clotting proteases, fXII, fX, fXII, plasmin, and plasma kallikrein alongside blood clotting regulatory serpins (antithrombin III and heparin cofactor II). Similarly, rIxsS17 interacted with both complement system serine proteases, C1s, C2, and factor I and the regulatory serpin, plasma protease C1 inhibitor. Consistently, we validated that rIxsS17 dose dependently blocked deposition of the complement membrane attack complex via the lectin complement pathway and protected complement sensitive B. burgdorferi from complement-mediated killing. Likewise, co-inoculating C3H/HeN mice with rIxsS17 and B. burgdorferi significantly enhanced colonization of mouse heart and skin organs in a reverse dose dependent manner. Taken together, our data suggests an important role for IxsS17 in tick feeding and B. burgdorferi colonization of the host.

Hypoxia-related carbonic anhydrase 9 induces serpinB9 expression in cancer cells and apoptosis in T cells via acidosis.

Hypoxia is a common feature of solid tumors. However, the impact of hypoxia on immune cells within tumor environments remains underexplored. Carbonic anhydrase 9 (CA9) is a hypoxia-responsive tumor-associated enzyme. We previously noted that regardless of human CA9 (hCA9) expression, hCA9-expressing mouse renal cell carcinoma RENCA (RENCA/hCA9) presented as a "cold" tumor in syngeneic aged mice. This study delves into the mechanisms behind this observation. Gene microarray analyses showed that RENCA/hCA9 cells exhibited elevated mouse serpinB9, an inhibitor of granzyme B, relative to RENCA cells. Corroborating this, RENCA/hCA9 cells displayed heightened resistance to antigen-specific cytotoxic T cells compared with RENCA cells. Notably, siRNA-mediated serpinB9 knockdown reclaimed this sensitivity. In vivo tests showed that serpinB9 inhibitor administration slowed RENCA tumor growth, but this effect was reduced in RENCA/hCA9 tumors, even with adjunctive immune checkpoint blockade therapy. Further, inducing hypoxia or introducing the mouse CA9 gene upregulated serpinB9 expression, and siRNA-mediated knockdown of the mouse CA9 gene inhibited the hypoxia-induced induction of serpinB9 in the original RENCA cells. Supernatants from RENCA/hCA9 cultures had lower pH than those from RENCA, suggesting acidosis. This acidity enhanced serpinB9 expression and T cell apoptosis. Moreover, coculturing with RENCA/hCA9 cells more actively prompted T cell apoptosis than with RENCA cells. Collectively, these findings suggest hypoxia-associated CA9 not only boosts serpinB9 in cancer cells but also synergistically intensifies T cell apoptosis via acidosis, characterizing RENCA/hCA9 tumors as "cold."

The Inhibition of Serine Proteases by Serpins Is Augmented by Negatively Charged Heparin: A Concise Review of Some Clinically Relevant Interactions.

Serine proteases are members of a large family of hydrolytic enzymes in which a particular serine residue in the active site performs an essential role as a nucleophile, which is required for their proteolytic cleavage function. The array of functions performed by serine proteases is vast and includes, among others, the following: (i) the ability to fight infections; (ii) the activation of blood coagulation or blood clot lysis systems; (iii) the activation of digestive enzymes; and (iv) reproduction. Serine protease activity is highly regulated by multiple families of protease inhibitors, known collectively as the SERine Protease INhibitor (SERPIN). The serpins use a conformational change mechanism to inhibit proteases in an irreversible way. The unusual conformational change required for serpin function provides an elegant opportunity for allosteric regulation by the binding of cofactors, of which the most well-studied is heparin. The goal of this review is to discuss some of the clinically relevant serine protease-serpin interactions that may be enhanced by heparin or other negatively charged polysaccharides. The paired serine protease-serpin in the framework of heparin that we review includes the following: thrombin-antithrombin III, plasmin-anti-plasmin, C1 esterase/kallikrein-C1 esterase inhibitor, and furin/TMPRSS2 (serine protease Transmembrane Protease 2)-alpha-1-antitrypsin, with the latter in the context of COVID-19 and prostate cancer.

Tissue-targeted and localized AAV5-DCN and AAV5-PEDF combination gene therapy abrogates corneal fibrosis and concurrent neovascularization in rabbit eyes in vivo.

PURPOSE: Corneal fibrosis and neovascularization (CNV) after ocular trauma impairs vision. This study tested therapeutic potential of tissue-targeted adeno-associated virus5 (AAV5) mediated decorin (DCN) and pigment epithelium-derived factor (PEDF) combination genes in vivo. METHODS: Corneal fibrosis and CNV were induced in New Zealand White rabbits via chemical trauma. Gene therapy in stroma was delivered 30-min after chemical-trauma via topical AAV5-DCN and AAV5-PEDF application using a cloning cylinder. Clinical eye examinations and multimodal imaging in live rabbits were performed periodically and corneal tissues were collected 9-day and 15-day post euthanasia. Histological, cellular, and molecular and apoptosis assays were used for efficacy, tolerability, and mechanistic studies. RESULTS: The AAV5-DCN and AAV5-PEDF combination gene therapy significantly reduced corneal fibrosis (p < 0.01 or p < 0.001) and CNV (p < 0.001) in therapy-given (chemical-trauma and AAV5-DCN + AAV5-PEDF) rabbit eyes compared to the no-therapy given eyes (chemical-trauma and AAV5-naked vector). Histopathological analyses demonstrated significantly reduced fibrotic α-smooth muscle actin and endothelial lectin expression in therapy-given corneas compared to no-therapy corneas on day-9 (p < 0.001) and day-15 (p < 0.001). Further, therapy-given corneas showed significantly increased Fas-ligand mRNA levels (p < 0.001) and apoptotic cell death in neovessels (p < 0.001) compared to no-therapy corneas. AAV5 delivered 2.69 × 107 copies of DCN and 2.31 × 107 copies of PEDF genes per µg of DNA. AAV5 vector and delivered DCN and PEDF genes found tolerable to the rabbit eyes and caused no significant toxicity to the cornea. CONCLUSION: The combination AAV5-DCN and AAV5-PEDF topical gene therapy effectively reduces corneal fibrosis and CNV with high tolerability in vivo in rabbits. Additional studies are warranted.

The Role of Galectin3, Tubulinß, and Maspin in Promoting Tumor Budding in Colorectal Carcinoma and Their Clinical Implications.

Colorectal cancer (CRC) is a leading cause of death worldwide. Despite the advances in surgical and therapeutic management, tumor metastases and poor prognosis are still major problems. Tumor budding is a relevant prognostic factor in CRC, and it can predict tumor metastasis. Galectin3 is responsible for the development and progression of many cancers through the regulation of cell-cell/cell-matrix interactions and tumor cell invasion. Tubulin is a microtubule protein, and maspin is a serine protease inhibitor; both induce tumor cell invasion through the stimulation of epithelial-mesenchymal transition. This study aims to evaluate the relationship between the expression of galecin3, tubulinß, and maspin in CRC and clinicopathological features, including tumor budding, their prognostic roles, and clinical implications using immunohistochemistry. Galectin3, tubulinß, and maspin were detected in tumor cells in 95%, 65%, and 87.5% of cases and in stromal cells in 28.8%, 40%, and 0% of cases. High expression of galectin3 and tubulinß expression either in tumor cells or stroma was significantly associated with aggressive tumor features such as lymph node metastasis, lymphovascular invasion, tumor budding, and advanced tumor stage. The nucleocytoplasmic expression of maspin in tumor cells showed a significant association with deeper tumor invasion, lymph node metastasis, tumor budding, and advanced tumor stage. Significant associations were found between high galectin3 tumor cell expression and nucleocytoplasmic maspin and shorter survival. High expression of galectin3, tubulinß, and nucleocytoplasmic maspin were significantly associated with aggressive tumor features such as tumor invasion, metastasis, high tumor budding, and short survival in CRC. They could be used as biomarkers for tumor budding and tumor aggressiveness in CRC and may be considered for future target therapy.

The identification of a serpin with immune defense role in oriental river prawn Macrobrachium nipponense.

Serpins are a protein superfamily of serine protease inhibitors. One of their functions is to participate in immune responses by inhibiting the activation of prophenoloxidase. To elucidate the immune role of serpin in Macrobrachium nipponense, a serpin gene (Mnserpin) was cloned from M. nipponense in this study. Mnserpin protein has an N-terminal signal peptide and a serpin domain that contains a hinge region, a signature sequence of serpin and a P1(arginine)-P1' scissile bond, and evolutionally closely related to the crustacean serpins. Mnserpin highly expressed in the hepatopancreas and gill. Mnserpin expression increased first and then decreased after Vibrio parahaemolyticus and Aeromonas hydrophila infection, and was knocked down by dsMnserpin injection with a maximum knockdown efficiency of 92 %. Mnserpin knockdown increased the expression of the clip domain serine protease and prophenoloxidase genes and phenoloxidase activity of M. nipponense as well as its mortality rate after V. parahaemolyticus and A. hydrophila infection. The recombinant Mnserpin (rMnserpin) showed bacteria-binding and bacteriostatic activity in vitro. Moreover, rMnserpin injection decreased the bacterial number and the mortality rate of M. nipponense post V. parahaemolyticus and A. hydrophila infection. These results suggested that Mnserpin plays a major role in the innate immune response of M. nipponense.

Mass Spectrometry-Based Proteomics Identifies Serpin B9 as a Key Protein in Promoting Bone Metastases in Lung Cancer.

Bone metastasis (BM) is one of the most common complications of advanced cancer. Immunotherapy for bone metastasis of lung cancer (LCBM) is not so promising and the immune mechanisms are still unknown. Here, we utilized a model of BM by injecting cancer cells through caudal artery (CA) to screen out a highly bone metastatic derivative (LLC1-BM3) from a murine lung cancer cell line LLC1. Mass spectrometry-based proteomics was performed in LLC1-parental and LLC1-BM3 cells. Combining with prognostic survival information from patients with lung cancer, we identified serpin B9 (SB9) as a key factor in BM. Molecular characterization showed that SB9 overexpression was associated with poor prognosis and high bone metastatic burden in lung cancer. Moreover, SB9 could increase the ability of lung cancer cells to metastasize to the bone. The mechanistic studies revealed that tumor-derived SB9 promoted BM through an immune cell-dependent way by inactivating granzyme B, manifesting with the decreased infiltration of cytotoxic T cells and increased expression level of exhausted markers. A specific SB9-targeting inhibitor [1,3-benzoxazole-6-carboxylic acid (BTCA)] significantly suppressed LCBM in the CA mouse model. This study reveals that SB9 may serve as a therapeutic target and potential prognostic marker for patients with LCBM. IMPLICATIONS: SB9 as a therapeutic target for LCBM.

Kallistatin as an inhibitory protein against colorectal cancer cells through binding to LRP6.

Kallistatin (KL) is a member of the serine proteinase inhibitor (serpin) family regulating oxidative stress, vascular relaxation, inflammation, angiogenesis, cell proliferation, and invasion. The heparin-binding site of Kallistatin has an important role in the interaction with LRP6 leading to the blockade of the Wnt signaling pathway. In this study, we aimed to explore the structural basis of the Kallistatin-LRP6E1E4 complex using in silico approaches and evaluating the anti-proliferative, apoptotic, and cell cycle arrest activities of Kallistatin in colon cancer lines. The molecular docking showed Kallistatin could bind to the LRP6E3E4 much stronger than LRP6E1E2. The Kallistatin-LRP6E1E2 and Kallistatin-LRP6E3E4 complexes were stable during Molecular Dynamics (MD) simulation. The Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) showed that the Kallistatin-LRP6E3E4 has a higher binding affinity compared to Kallistatin-LRP6E1E2. Kallistatin induced higher cytotoxicity and apoptosis in HCT116 compared to the SW480 cell line. This protein-induced cell-cycle arrest in both cell lines at the G1 phase. The B-catenin, cyclin D1, and c-Myc expression levels were decreased in response to treatment with Kallistatin in both cell lines while the LRP6 expression level was decreased in the HCT116 cell line. Kallistatin has a greater effect on the HCT116 cell line compared to the SW480 cell line. Kallistatin can be used as a cytotoxic and apoptotic-inducing agent in colorectal cancer cell lines.

LAMB3 Promotes Intestinal Inflammation Through SERPINA3 and Is Directly Transcriptionally Regulated by P65 in Inflammatory Bowel Disease.

BACKGROUND: Various extracellular matrix (ECM) reshaping events are involved in inflammatory bowel disease (IBD). LAMB3 is a vital subunit of laminin-332, an important ECM component. Data on the biological function of LAMB3 in intestinal inflammation are lacking. Our aim is to discuss the effect of LAMB3 in IBD. METHODS: LAMB3 expression was assessed in cultured intestinal epithelial cells, inflamed mucosal tissues of patients and mouse colitis models. RNA sequencing, quantitative real-time polymerase chain reaction and Western blotting were used to detect the LAMB3 expression distribution and potential downstream target genes. Dual-luciferase assays and chromatin immunoprecipitation-quantitative polymerase chain reaction were used to determine whether P65 could transcriptionally activate LAMB3 under tumor necrosis factor α stimulation. RESULTS: LAMB3 expression was increased in inflammatory states in intestinal epithelial cells and colonoids and was associated with adverse clinical outcomes in Crohn's disease. Knockdown of LAMB3 inhibited the expression of proinflammatory cytokines. Mechanistically, LAMB3 expression was directly transcriptionally activated by P65 and was inhibited by nuclear factor kappa B inhibitors under tumor necrosis factor α stimulation. Furthermore, RNA sequencing and replenishment experiments revealed that LAMB3 upregulated SERPINA3 to promote intestinal inflammation via the integrin α3ß1/FAK pathway. CONCLUSION: We propose that LAMB3 could serve as a potential therapeutic target of IBD and a predictor of intestinal stenosis of Crohn's disease. Our findings demonstrate the important role of ECM in the progression of IBD and offer an experimental basis for the treatment and prognosis of IBD.

Short peptides derived from pigment epithelium-derived factor attenuate retinal ischemia reperfusion injury through inhibition of apoptosis and inflammatory response in rats.

Pigment epithelium-derived factor (PEDF) is widely recognized as a neuroprotective factor expressed in the retina and has shown therapeutic potential in several retinal diseases. Our study aimed to identify the neuroprotective fragment in PEDF and investigate its protective activity in retinas under ischemia-reperfusion (IR) condition. We synthesized a series of shorter synthetic peptides, 6-mer (Ser93-Gln98) and its d-form variant (6 dS) derived from the 44-mer (Val78-Thr121; a PEDF neurotrophic fragment), to determine their cytoprotective activity in IR injury, which was induced in rat retinas by injection of saline into the anterior chamber to increase the intraocular pressure (IOP) followed by reperfusion. We found the cytoprotective effect of 6-mer on glutamate-treated Neuro-2a cells and tert-butyl hydroperoxide (tBHP)-treated 661W cells were 2.6-fold and 1.5-fold higher than the 44-mer, respectively. The cytoprotective effect was blocked by a chemical inhibitor atglistatin and blocking antibody targeting PEDF receptor (PEDF-R). IR induced several impairments in retina, including cell apoptosis, activation of microglia/macroglia, degeneration of retinal capillaries, reduction in electroretinography (ERG) amplitudes, and retinal atrophy. Such IR injuries were ameliorated by treatment with 6-mer and 6 dS eye drops. Also, the neuroprotective activity of 6-mer and 6 dS in ischemic retinas were dramatically reversed by atglistatin preconditioning. Taken together, our data demonstrate smallest neuroprotective fragment of PEDF has potential to treat retinal degeneration-related diseases.

Hypothermic machine perfusion reduces donation after circulatory death liver ischemia-reperfusion injury through the SERPINA3-mediated PI3Kδ/Akt pathway.

Hypothermic machine perfusion (HMP) has been demonstrated to be more effective in mitigating ischemia-reperfusion injury (IRI) of donation after circulatory death (DCD) organs than cold storage (CS), yet the underlying mechanism remains obscure. We aimed to propose a novel therapeutic approach to ameliorate IRI in DCD liver transplantation. Twelve clinical liver samples were randomly assigned to HMP or CS treatment and subsequent transcriptomics analysis was performed. By combining in vivo HMP models, we discovered that HMP attenuated inflammation, oxidative stress, and apoptosis in DCD liver through a SEPRINA3-mediated PI3Kδ/AKT signaling cascade. Moreover, in the hypoxia/reoxygenation (H/R) model of BRL-3A, overexpression of SERPINA3 mitigated H/R-induced apoptosis, while SERPINA3 knockdown exacerbated cell injury. Idelalisib (IDE) treatment also reversed the protective effect of SERPINA3 overexpression. Overall, our research provided new insights into therapeutic strategies and identified potential novel molecular targets for therapeutic intervention against DCD liver.

Serpin family E member 1 enhances myometrium contractility by increasing ATP production during labor.

The uterine contraction during labor, a process with repetitive hypoxia and high energy consumption, is essential for successful delivery. However, the molecular mechanism of myometrial contraction regulation is unknown. Serpin family E member 1 (SERPINE1), one of the most upregulated genes in laboring myometrium in both transcriptome and proteome, was highlighted in our previous study. Here, we confirmed SERPINE1 is upregulated in myometrium during labor. Blockade of SERPINE1 using small interfering RNA (siRNA) or inhibitor (Tiplaxtinin) under hypoxic conditions in myocytes or myometrium in vitro showed a decrease contractility, which was achieved by regulating ATP production. Chromatin immunoprecipitation (ChIP-seq), Co-immunoprecipitation (Co-IP), and glutathione-S-transferase (GST) pull down explored that the promoter of SERPINE1 is directly activated by hypoxia-inducible factor-1α (HIF-1α) and SERPINE1 interacts with ATP Synthase Peripheral Stalk Subunit F6 (ATP5PF). Together they enhance hypoxia driven myometrial contraction by maintaining ATP production in the key oxidative phosphorylation pathway. The results provide new insight for uterine contraction regulation, and potential novel therapeutic targets for labor management.

CTSC promoted the migration and invasion of glioma cells via activation of STAT3/SERPINA3 axis.

Cathepsin C (CTSC) has been reported to be upregulated in several cancers, however, there are still many missing links about the role of CTSC in glioma. To address this knowledge gap, the present study employed bioinformatics analysis, Transwell assay, RT-qPCR and Western blot assays to investigate the expression level of CTSC in glioma tissues, its relationship with survival period, and its effect on the migration and invasion ability of glioma cells. The findings revealed that CTSC was upregulated in glioma and was associated with poor prognosis. Moreover, CTSC was found to promote cell migration and invasion abilities as well as epithelial-mesenchymal transition (EMT). A further study found that CTSC induced SERPINA3 and STAT3 expression in glioma cells. Additionally, we demonstrated that STAT3 signaling mediated upregulation of SERPINA3 expression by CTSC. In sum, our findings suggest that CTSC activates the STAT3/SERPINA3 axis to promote migration and invasion of glioma cells, which may lead to new potential therapeutic approaches for humans with cancer.

Unveiling the role of miR-137-3p/miR-296-5p/SERPINA3 signaling in colorectal cancer progression: integrative analysis of gene expression profiles and in vitro studies.

BACKGROUND: Colorectal cancer (CRC) is a prevalent malignancy worldwide, with increasing incidence and mortality rates. Although treatment options have improved, CRC remains a leading cause of death due to metastasis. Early intervention can significantly improve patient outcomes, making it crucial to understand the molecular mechanisms underlying CRC metastasis. In this study, we performed bioinformatics analysis to identify potential genes associated with CRC metastasis. METHODS: We downloaded and integrated gene expression datasets (GSE89393, GSE100243, and GSE144259) from GEO database. Differential expression analysis was conducted, followed by Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The hub gene SERPINA3 was selected for further in vitro functional studies. Additionally, the role of miR-137-3p/miR-296-5p/ Serpin family A member 3 (SERPINA3) in CRC cell function was investigated using in vitro assays. RESULTS: Analysis of the gene expression datasets revealed differentially expressed genes (DEGs) associated with CRC metastasis. GO analysis showed enrichment in biological processes such as blood coagulation regulation and wound healing. Cellular component analysis highlighted extracellular matrix components and secretory granules. Molecular function analysis identified activities such as serine-type endopeptidase inhibition and lipoprotein receptor binding. KEGG analysis revealed involvement in pathways related to complement and coagulation cascades, cholesterol metabolism, and immune responses. The common DEGs among the datasets were further investigated. We identified SERPINA3 as a hub gene associated with CRC metastasis. SERPINA3 exerted enhanced effects on migration, proliferation and epithelial-mesenchymal transition (EMT) and inhibitory effects on caspase-3/-9 activities in HT29 and SW620 cells. MiR-137-3p overexpression increased activities of caspase-3/-9, decreased migration and proliferation, and also repressed EMT in HT29 cells, which were obviously attenuated by SERPINA3 enforced overexpression. Consistently, SERPINA3 enforced overexpression also largely reversed miR-296-5p mimics-induced increased in activities of caspase-3/-9, decrease in migration, proliferation and EMT in HT29 cells. CONCLUSION: Through bioinformatics analysis, we identified potential genes associated with CRC metastasis. The functional studies focusing on SERPINA3/miR-137-3p/miR-296-5p further consolidated its role in regulating CRC progression. Our findings provide insights into novel mechanisms underlying CRC metastasis and might contribute to the development of effective treatment strategies. However, the role of SERPINA3/miR-137-3p/miR-296-5p signaling in CRC still requires further investigation.