Transferase dUTP Nick End Labeling Research Articles

P30 Exposure to particulate matter induces structural and pigmentary changes to human skin

Abstract Introduction and aims The majority of the world’s population reside in places with high levels of air pollution. The impact of air pollution on human health has been extensively studied and demonstrates a significant correlation between particulate matter (PM) and risk of cardiovascular and respiratory diseases. However, little is known about the impact of air pollution on human skin health. Here, we develop an ex vivo skin model to investigate cutaneous changes induced by the topical application of PM. Methods PM (0, 0.5, 3, 6, 10, 20 and 40 μg mL−1) was topically applied to Genoskin® NativeSkin Access® (Genoskin, Salem, MA, USA) human ex vivo skin samples of White European ancestry (n = 3) for 8 days, with reapplication on day 4. Resultant samples were assessed using haematoxylin and eosin staining (for morphometric analyses), immunofluorescence for Ki67 and terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) (to determine cellular turnover), while pigmentary changes were analysed by colourimetry, Warthin–Starry (WS) staining and immunohistochemistry for the melanin markers tyrosinase (TYR), gp100 and c-kit. Results Morphological assessment revealed that epidermal thickness was significantly reduced following exposure to higher doses of PM (r = −0.9163, P < 0.01). In addition, there was reduced epidermal cell proliferation (Ki67) and increased apoptosis (TUNEL) at higher doses of PM. Exposure to PM resulted in a dose-dependent decrease in individual typology angle, which is indicative of skin colour becoming darker (r = −0.7895, P < 0.05). Furthermore, WS staining demonstrated significantly increased epidermal melanin (r = 0.9583, P < 0.01) and significantly increased expression of melanin markers TYR (P < 0.01), gp100 (P < 0.001) and c-kit (P < 0.01) in response to PM exposure. Conclusions This study confirms that PM induces pigmentary changes to ex vivo human skin by increasing epidermal melanin. Increased expression of c-kit in response to PM exposure suggests that increased pigmentation occurs owing to the upregulation of the microphthalmia-associated transcription factor signalling pathway. Further investigation of the mechanistic drivers of increased pigmentation is warranted.

P31 Modelling epidermal melanocytes behaviour during plaque psoriasis resolution

Abstract Introduction and aims Melanocytes in the human epidermis exist in the stratum basale at a ratio of 1 melanocyte to approximately 10 keratinocytes. They do not divide, even upon ultraviolet radiation stimulation, and do not appear to apoptose. In psoriasis, this melanocyte/keratinocyte ratio is retained, despite the enormous keratinocyte hyperplasia, inferring a commensurate expansion in melanocyte number. Therefore, here we investigate whether melanocyte number is altered following the reduction of keratinocytes upon treatment of psoriasis plaques with biologic therapy. Methods Matching lesional and nonlesional epidermis sets of patients (n = 4) at day 0 and after 4 and 12 weeks of treatment were compared with healthy controls. To assess the presence of apoptotic melanocytes, the terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay was performed. Immunohistochemistry with microphthalmia-associated transcription factor (melanocyte marker) and Ki-67 (proliferation marker) was used to assess melanocyte proliferation. Finally, melanocyte number per mm of basal layer and melanocyte/keratinocyte ratio were estimated by image analysis using ImageJ. Results Proliferating melanocytes were detected in nonlesional and lesional samples and in resolving plaques after 4 and 12 weeks of treatment but not in healthy epidermis. Melanocyte number per mm of basal layer was significantly higher in nonlesional than lesional sections compared with healthy controls (P < 0.01) suggesting that melanocyte expansion already occurs prior to lesion establishment, revealing early melanocyte deregulation. After 4 and 12 weeks of treatment the number of melanocytes in lesional and nonlesional sections was still significantly higher than controls (P < 0.05) and had not returned to normal despite the dramatic reduction in keratinocytes. The TUNEL assay did not detect evidence of melanocyte apoptosis. Conclusions This study shows that rare melanocyte proliferation occurs in nonlesional psoriasis skin and in resolving lesional skin. Given our inability to detect apoptotic melanocytes, it remains unclear how melanocyte numbers return to normal after plaque resolution These unique findings support a role for melanocytes in psoriasis pathogenesis and encourage further investigation.

ANO6 (TMEM16F) inhibits gastrointestinal stromal tumor growth and induces ferroptosis.

Herein, we elucidate the potential role of ANO6 (TMEM16F) in gastrointestinal stromal tumors (GISTs). ANO6 expression in GIST and adjacent normal tissues was determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. Cell proliferation, apoptosis, and pyroptosis were examined utilizing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, terminal deoxynucleotidyl transferase dUTP Nick-End Labeling staining, and flow cytometry. In addition, the total iron and Fe2+ levels were assessed. IL-18 and IL-1β levels were also evaluated. Lipid reactive oxygen species (ROS), cystine (Cys), glutathione (GSH), and glutathione peroxidase 4 (GPX4) levels were evaluated using appropriate kits. Ferroptotic markers, including Ptgs2, Chac1, SLC7A11, and SLC3A2, were analyzed by RT-qPCR, western blotting, and immunohistochemistry. ANO6 expression decreased in GIST tissues. ANO6-plasmid inhibits proliferation, induces apoptosis, and promotes pyroptosis in GIST-T1 and GIST-T1 IR cells. The ANO6-plasmid induced ferroptosis, as confirmed by enhanced lipid ROS levels, increased intracellular concentrations of total iron and Fe2+, promoted Ptgs2 and Chac1 expression, reduced Cys, GSH, and GPX4 levels, and downregulated SLC7A11 and SLC3A2 expression after in vitro and in vivo treatment with ANO6-plasmid. Moreover, the ANO6-plasmid inhibited GIST growth in vivo. Therefore, ANO6 may be a promising therapeutic target for blocking the development of GIST via the induction of apoptosis, pyroptosis, and ferroptosis.

Successful Extension of Vascularized Composite Allograft Perfusion Cold Storage to 24 h in a Rat Hindlimb Transplant Model.

Vascularized composite allograft transplantation is a treatment option for complex tissue injuries; however, ischemia reperfusion injury and high acute rejection rates remain a challenge. Hypothermic machine perfusion using acellular storage perfusate is a potential solution. This study evaluated the University of Wisconsin Kidney Preservation Solution-1 (KPS-1) compared with normal saline (NS) for preservation of donor rat hindlimbs subjected to 24 h of ex vivo perfusion cold storage. Hindlimbs were subjected to 24-h perfusion cold storage with heparinized KPS-1 (n = 6) or heparinized NS (n = 6). Flow, resistance, and pH were measured continuously. At the end of the 24-h period, tissue was collected for histological analysis of edema and apoptosis. KPS-1 perfused limbs showed significantly less edema than the NS group, as evidenced by lower limb weight gain (P < 0.001) and less interfascicular space (P < 0.001). KPS-perfused muscle had significantly less cell death than NS-perfused muscle based on terminal deoxynucleotidyl transferase dUTP nick-end labeling (P < 0.001) and cleaved caspase-3 staining (P = 0.045). During hypothermic machine perfusion, a significant decrease in pH over time was detected in both groups, with a significantly greater decline in pH in the KPS-1 group than in the NS group. There were no significant differences overall and over time in flow rate or vascular resistance between the KPS and NS groups. Perfusion with KPS-1 can successfully extend vascularized composite allograft perfusion cold storage for 24 h in a rat hindlimb model without significant edema or cell death.

Neuroprotective effects of apigenin on retinal ganglion cells in ischemia/reperfusion: modulating mitochondrial dynamics in in vivo and in vitro models

BackgroundRetinal ischemia/reperfusion (RIR) is implicated in various forms of optic neuropathies, yet effective treatments are lacking. RIR leads to the death of retinal ganglion cells (RGCs) and subsequent vision loss, posing detrimental effects on both physical and mental health. Apigenin (API), derived from a wide range of sources, has been reported to exert protective effects against ischemia/reperfusion injuries in various organs, such as the brain, kidney, myocardium, and liver. In this study, we investigated the protective effect of API and its underlying mechanisms on RGC degeneration induced by retinal ischemia/reperfusion (RIR).MethodsAn in vivo model was induced by anterior chamber perfusion following intravitreal injection of API one day prior to the procedure. Meanwhile, an in vitro model was established through 1% oxygen and glucose deprivation. The neuroprotective effects of API were evaluated using H&E staining, spectral-domain optical coherence tomography (SD-OCT), Fluoro-Gold retrograde labeling, and Photopic negative response (PhNR). Furthermore, transmission electron microscopy (TEM) was employed to observe mitochondrial crista morphology and integrity. To elucidate the underlying mechanisms of API, the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, flow cytometry assay, western blot, cell counting kit-8 (CCK-8) assay, lactate dehydrogenase (LDH) assay, JC-1 kit assay, dichlorofluorescein-diacetate (DCFH-DA) assay, as well as TMRE and Mito-tracker staining were conducted.ResultsAPI treatment protected retinal inner plexiform layer (IPL) and ganglion cell complex (GCC), and improved the function of retinal ganglion cells (RGCs). Additionally, API reduced RGC apoptosis and decreased lactate dehydrogenase (LDH) release by upregulating Bcl-2 and Bcl-xL expression, while downregulating Bax and cleaved caspase-3 expression. Furthermore, API increased mitochondrial membrane potential (MMP) and decreased extracellular reactive oxygen species (ROS) production. These effects were achieved by enhancing mitochondrial function, restoring mitochondrial cristae morphology and integrity, and regulating the expression of OPA1, MFN2, and DRP1, thereby regulating mitochondrial dynamics involving fusion and fission.ConclusionAPI protects RGCs against RIR injury by modulating mitochondrial dynamics, promoting mitochondrial fusion and fission.

Gypenoside XLIX Activates the Sirt1/Nrf2 Signaling Pathway to Inhibit NLRP3 Inflammasome Activation to Alleviate Septic Acute Lung Injury.

Currently, treatment options for acute lung injury (ALI) are limited. Gypenoside XLIX (Gyp-XLIX) is known for its anti-inflammatory properties, but there is a lack of extensive research on its effects against ALI. This study induced ALI in mice through cecal ligation and puncture surgery and investigated the biological activity and potential mechanisms of Gypenoside XLIX (40mg/kg) by intraperitoneal injection. The in vitro ALI model was established using mouse lung epithelial (MLE-12) cells stimulated with lipopolysaccharide (LPS) and adenosine triphosphate (ATP). Various methods, including Hematoxylin and Eosin (H&E) staining, biochemical assay kits, Quantitative Polymerase Chain Reaction (qPCR) analysis, Western blotting, Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) assay, immunofluorescence, and flow cytometry, were employed for this research. The results indicated that pretreatment with Gypenoside XLIX significantly alleviated pathological damage in mouse lung tissues and reduced the expression levels of inflammatory factors. Additionally, Gypenoside XLIX inhibited ROS levels and NLRP3 inflammasome, possibly mediated by the Sirt1/Nrf2 signaling pathway. Moreover, Gypenoside XLIX significantly inhibited sepsis-induced lung cell apoptosis and excessive autophagy of mitochondria. Specifically, it suppressed mitochondrial pathway apoptosis and the Pink1/Parkin pathway of mitochondrial autophagy. These findings reveal the multifaceted effects of Gypenoside XLIX in anti-inflammatory, antioxidative, and inhibition of cell apoptosis and autophagy. This provides strong support for its therapeutic potential in sepsis-related lung injuries.

Salidroside ameliorates acute liver transplantation rejection in rats by inhibiting neutrophil extracellular trap formation.

Acute rejection is an important factor affecting the survival of recipients after liver transplantation. Salidroside has various properties, including anti-inflammatory, antioxidant, and hepatoprotective properties. This study aims to investigate whether salidroside can prevent acute rejection after liver transplantation and to examine the underlying mechanisms involved. An in vivo acute rejection model is established in rats that are pretreated with tacrolimus (1 mg/kg/d) or salidroside (10 or 20 mg/kg/d) for seven days after liver transplantation. In addition, an in vitro experiment is performed using neutrophils incubated with salidroside (1, 10, 50 or 100 μM). Hematoxylin-eosin staining, terminal deoxynucleotidyl transferase dUTP nick-end labeling staining, immunosorbent assays, immunofluorescence analysis, Evans blue staining, and western blot analysis are performed to examine the impact of salidroside on NET formation and acute rejection in vitro and in vivo. We find that Salidroside treatment reduces pathological liver damage, serum aminotransferase level, and serum levels of IL-1β, IL-6, and TNF-α in vivo. The expressions of proteins associated with the HMGB1/TLR-4/MAPK signaling pathway (HMGB1, TLR-4, p-ERK1/2, p-JNK, p-P38, cleaved caspase-3, cleaved caspase-9, Bcl-2, Bax, IL-1β, TNF-α, and IL-6) are also decreased after salidroside treatment. In vitro experiments show that the release of HMGB1/TLR-4/MAPK signaling pathway-associated proteins from neutrophils treated with lipopolysaccharide is decreased by salidroside. Moreover, salidroside inhibits NETosis and protects against acute rejection by regulating the HMGB1/TLR-4/MAPK signaling pathway. Furthermore, salidroside combined with tacrolimus has a better effect than either of the other treatments alone. In summary, salidroside can prevent acute liver rejection after liver transplantation by reducing neutrophil extracellular trap development through the HMGB1/TLR-4/MAPK signaling pathway.

In Vitro Effects of Boric Acid on Cell Cycle, Apoptosis, and miRNAs in Medullary Thyroid Cancer Cells.

Medullary thyroid cancer (MTC) is a highly aggressive and chemotherapy-resistant cancer originating from the thyroid's parafollicular C cells. Due to its resistance to conventional treatments, alternative therapies such as boric acid have been explored. Boric acid, a boron-based compound, has shown anticarcinogenic effects, positioning it as a potential treatment option for MTC. TT medullary thyroid carcinoma cell line (TT cells) and human thyroid fibroblast (HThF cells) were utilized for the cell culture experiments. Cell viability was assessed using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay. Total RNA was extracted using Trizol reagent for gene expression and microRNA (miRNA) analysis via reverse transcription-polymerase chain reaction (RT-PCR). The extent of apoptosis induced by boric acid was determined using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Colony formation assays were conducted to evaluate the impact of boric acid on the colony-forming ability of MTC cells. At 48h, 50% inhibitory concentration (IC50) of boric acid was found to be 35μM. Treatment with boric acid resulted in significant modulation of apoptosis-related genes and miRNAs, including increased expression of phorbol-12-myristate-13-acetate-induced protein 1(NOXA), apoptotic protease activating factor 1 (APAF-1), Bcl-2-associated X protein (Bax), caspase-3, and caspase-9. In contrast, the expression of B cell lymphoma 2 (Bcl2), B cell lymphoma- extra-large (Bcl-xl), and microRNA-21 (miR-21), which are linked to the aggressiveness of MTC, was significantly reduced. The TUNEL assay indicated a 14% apoptosis rate, and there was a 67.9% reduction in colony formation, as shown by the colony formation assay. Our study suggests that boric acid may have anticancer activity in MTC by modulating apoptotic pathways. These findings suggest that boric acid could be a potential therapeutic agent for MTC and possibly for other malignancies with similar pathogenic mechanisms.

A recombinant plasmid encoding human hepatocyte growth factor promotes healing of combined radiation-trauma skin injury involved in regulating Nrf2 pathway in mice.

Combined radiation-trauma skin injury represents a severe and intractable condition that urgently requires effective therapeutic interventions. In this context, hepatocyte growth factor (HGF), a multifunctional growth factor with regulating cell survival, angiogenesis, anti-inflammation and antioxidation, may be valuable for the treatment of combined radiation-trauma injury. This study investigated the protective effects of a recombinant plasmid encoding human HGF (pHGF) on irradiated human immortalized keratinocytes (HaCaT) cells in vitro, and its capability to promote the healing of combined radiation-trauma injuries in mice. The pHGF radioprotection on irradiated HaCaT cells in vitro was assessed by cell viability, the expression of Nrf2, Bcl-2 and Bax, as well as the secretion of inflammatory cytokines. In vivo therapeutic treatment, the irradiated mice with full-thickness skin wounds received pHGF local injection. The injuries were appraised based on relative wound area, pathology, immunohistochemical detection, terminal deoxynucleotidyl transferase dUTP nick end labelling assay and cytokine content. The transfection of pHGF increased the cell viability and Nrf2 expression in irradiated HaCaT cells. pHGF also significantly upregulated Bcl-2 expression, decreased the Bax/Bcl-2 ratio and inhibited the expression of interleukin-1β and tumor necrosis factor-α in irradiated cells. Local pHGF injection in vivo caused high HGF protein expression and noticeable accelerated healing of combined radiation-trauma injury. Moreover, pHGF administration upregulated Nrf2, vascular endothelial growth factor, Bcl-2 expression, downregulated Bax expression and mitigated inflammatory response. In conclusion, the protective effect of pHGF may be related to inhibiting apoptosis and inflammation involving by upregulating Nrf2. Local pHGF injection distinctly promoted the healing of combined radiation-trauma injury and demonstrates potential as a gene therapy intervention for combined radiation-trauma injury in clinic.

Sperm Chromatin Dispersion Test Detects Sperm DNA Fragmentation Mainly Associated with Unviable Spermatozoa and Underestimates the Values with Respect to TUNEL Assay.

Several clinical laboratories assess sperm DNA fragmentation (sDF) in addition to semen analysis in male infertility diagnosis. Among tests evaluating sDF, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) and SCD (Sperm Chromatin Dispersion) are widely used. Our lab developed a modified version of TUNEL (TUNEL/PI) able to distinguish two sperm populations (PI Brighter and PI Dimmer) differently associated with sperm viability and reproductive outcomes. The aim of this study was to compare sDF levels detected by SCD and TUNEL/PI in the semen samples from 71 male subjects attending our Andrology Laboratory. Our results demonstrate that SCD is less sensitive in determining sDF compared to TUNEL/PI. The statistically significant positive correlation found between sDF evaluated by SCD and PI Dimmer (consisting of all dead spermatozoa) suggests that SCD mainly detects sDF in unviable spermatozoa. We confirmed that most spermatozoa detected by SCD are unviable by performing SCD after incubation in hypo-osmotic medium to discriminate viable and unviable cells in 52 samples. Such results might explain the lower ability of this test in discriminating couples having successful ART outcomes demonstrated in published metanalyses. Overall, our results indicate that SCD is less sensitive in evaluating sDF for diagnostic purposes.

Role of cyclophilin A in aggravation of myocardial ischemia reperfusion injury via regulation of apoptosis mediated by thioredoxin-interacting protein.

The progression and persistence of myocardial ischemia/reperfusion injury (MI/RI) are strongly linked to local inflammatory responses and oxidative stress. Cyclophilin A (CypA), a pro-inflammatory factor, is involved in various cardiovascular diseases. However, the role and mechanism of action of CypA in MI/RI are still not fully understood. We used the Gene Expression Omnibus (GEO) database for bioinformatic analysis. We collected blood samples from patients and controls for detecting the levels of serum CypA using enzyme-linked immunosorbent assay (ELISA) kits. We then developed a myocardial ischemia/reperfusion (I/R) injury model in wild-type (WT) mice and Ppia-/- mice. We utilized echocardiography, hemodynamic measurements, hematoxylin and eosin (H&E) staining, immunohistochemistry, enzyme-linked immunosorbent assay, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining to determine the role of CypA in myocardial I/R injury. Finally, we conducted an in vitrostudy, cell transfection, flow cytometry, RNA interference, and a co-immunoprecipitation assay to clarify the mechanism of CypA in aggravating cardiomyocyte apoptosis. We found that CypA inhibited TXNIP degradation to enhance oxidative stress-induced cardiomyocyte apoptosis during MI/RI. By comparing and analyzing CypA expression in patients with coronary atherosclerotic heart disease and in healthy controls, we found that CypA was upregulated in patients with Coronary Atmospheric Heart Disease, and its expression was positively correlated with Gensini scores. In addition, CypA deficiency decreased cytokine expression, oxidative stress, and cardiomyocyte apoptosis in I/R-treated mice, eventually alleviating cardiac dysfunction. CypA knockdown also reduced H2O2-induced apoptosis in H9c2 cells. Mechanistically, we found that CypA inhibited K48-linked ubiquitination mediated by atrophin-interacting protein 4 (AIP4) and proteasomal degradation of TXNIP, a thioredoxin-binding protein that mediates oxidative stress and induces apoptosis. These findings highlight the critical role CypA plays in myocardial injury caused by oxidative stress-induced apoptosis, indicating that CypA can be a viable biomarker and a therapeutic target candidate for MI/RI.

Loganin exerts neuroprotective effect by inhibiting neuronal pyroptosis in rat with cerebral haemorrhage.

Intracerebral haemorrhage (ICH) presents significant challenges in clinical management because of the high morbidity and mortality, necessitating novel therapeutic approaches. This study aimed to assess the neuroprotective effects of loganin in a rat ICH model. Sprague-Dawley rats were used, subjected to a collagenase-induced ICH model, followed by loganin treatment at doses of 2.5, 5 and 10 mg/kg. Neurological functions were evaluated using the modified neurological severity score (mNSS) and a rotarod test. Results indicated a significant improvement in neurological functions in loganin-treated groups, evident from the mNSS and rotarod tests, suggesting dose-dependent neuroprotection. Loganin also effectively reduced the blood-brain barrier (BBB) permeability and cerebral oedema. Additionally, it mitigated cellular pyroptosis, as shown by terminal deoxynucleotidyl transferase dUTP nick-end labelling staining and western blot analysis, which indicated reduced levels of pyroptosis markers in treated rats. Furthermore, loganin's regulatory effects on the adenosine A2A receptor and myosin light chain kinase pathways were observed, potentially underpinning its protective mechanism against ICH. The study concludes that loganin exhibits significant neuroprotective properties in a rat ICH model, highlighting its potential as a novel therapeutic strategy. Despite promising results, the study needs further research to determine loganin's therapeutic potential in human ICH patients. This research paves the way for further exploration into loganin's clinical applications, potentially revolutionizing treatment strategies for patients suffering from intracerebral haemorrhage.

No side effects on rabbit retina or vitreous microenvironment by nd:YAG laser vitreolysis.

To explore the safety of Neodymium:Yttrium-aluminum-garnet (Nd:YAG) laser vitreolysis based on the histological examination of the retina and the alteration of vitreous cytokines in the rabbits. Nine male New Zealand rabbits underwent Nd:YAG laser vitreolysis of 10mJ x 500 pulses in the left eyes, while the right eyes were used as controls. Intraocular pressure, color fundus photography, and ultrasound B scan were measured before, as well as 1day, 4 weeks, and 12 weeks after Nd:YAG laser vitreolysis. Three rabbits were euthanized 1day, 4 weeks, and 12 weeks after treatment, respectively. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and hematoxylin-eosin (H&E) staining were used to look for pathological changes in the retina. An enzyme-linked immunosorbent assay (ELISA) was utilized to detect the expression of vascular endothelial growth factor (VEGF) and some inflammatory cytokines, including interferon inducible protein 10 (IP-10), monocyte chemoattractant protein 1 (MCP-1) and interlenkin 6 (IL-6) in the vitreous humor. The ascorbic acid (AsA) and total reactive antioxidant potential (TRAP) in the vitreous humor were also measured. Following Nd:YAG laser vitreolysis, the levels of VEGF, IP-10, MCP-1, IL6, AsA, and TRAP in the vitreous humor did not change substantially (P > 0.05). There were no detectable pathological changes in the retinal tissues, and no apoptotic signal was found. Rabbits tolerate Nd:YAG laser vitreolysis without observable impact on retinal tissue or the microenvironment of the vitreous.

Involvement of necroptotic cell death in macrophages in progression of bleomycin and LPS-induced acute exacerbation of idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is the severe form of interstitial pneumonias. Acute exacerbation (AE) of IPF is characterized by progressive lung fibrosis with the irreversible lung function decline and inflammation, and is often fatal with poor prognosis. However, the physiological and molecular mechanisms in AE of IPF are still not fully understood. In this study, we investigated the mechanism underlying AE of IPF, using bleomycin (BLM) and lipopolysaccharide (LPS) (BLM + LPS)-treated mice. The mice were treated with a single dose of 1.5 mg/kg BLM (on day 0) and/or 0.5 mg/kg LPS (on day 14), and maintained for another 7 days (total 21 days). Administration of BLM + LPS more severely aggravated the respiratory function, fibrosis, and inflammation in the lungs, together with the elevated interleukin-6 level in bronchoalveolar lavage fluid, than the control or BLM alone-treated mice. Moreover, the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay demonstrated that subsequent treatment with LPS elevated cell death in the lungs of BLM-administered mice. Furthermore, the expression levels of mixed lineage kinase domain-like protein (MLKL), a marker of necroptotic cell death, and CD68-positive macrophages were increased, and most of them were co-stained in the lungs of BLM + LPS-treated mice. These results, taken together, indicate that BLM + LPS treatment showed more exacerbated the respiratory function with extensive fibrosis and inflammation than treatment with BLM alone in mice. Fibrosis and inflammation in AE of IPF seen in BLM + LPS-administered mice included an increase in macrophages and their necroptotic cell death.

Succinic Acid Ameliorates Concanavalin A-Induced Hepatitis by Altering the Inflammatory Microenvironment and Expression of BCL-2 Family Proteins.

Autoimmune hepatitis (AIH) is a severe immune-mediated inflammatory liver disease that currently lacks feasible drug treatment methods. Our study aimed to evaluate the protective effect of succinic acid against AIH and provide a reliable method for the clinical treatment of AIH. We performed an in vivo study of the effects of succinic acid on concanavalin A (ConA)-induced liver injury in mice. We examined liver transaminase levels, performed hematoxylin and eosin (HE) staining, and observed apoptotic phenotypes in mice. We performed flow cytometry to detect changes in the number of neutrophils and monocytes, and used liposomes to eliminate the liver Kupffer cells and evaluate their role. We performed bioinformatics analysis, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and western blotting to detect mitochondrial apoptosis-induced changes in proteins from the B-cell lymphoma 2(Bcl-2) family. Succinic acid ameliorated ConA-induced AIH in a concentration-dependent manner, as reflected in the survival curve. HE and TUNEL staining and terminal deoxynucleotidyl transferase dUTP nick end labeling revealed decreased alanine transaminase and aspartate aminotransferase levels, and reduced liver inflammation and apoptosis. RT-qPCR and enzyme-linked immunosorbent assay revealed that succinic acid significantly reduced liver pro-inflammatory cytokine levels. Flow cytometry revealed significantly decreased levels of liver neutrophils. Moreover, the protective effect of succinic acid disappeared after the Kupffer cells were eliminated, confirming their important role in the effect. Bioinformatics analysis, RT-qPCR, and western blotting showed that succinic acid-induced changes in proteins from the Bcl-2 family involved mitochondrial apoptosis, indicating the molecular mechanism underlying the protective effect of succinic acid. Succinic acid ameliorated ConA-induced liver injury by regulating immune balance, inhibiting pro-inflammatory factors, and promoting anti-apoptotic proteins in the liver. This study provides novel insights into the biological functions and therapeutic potential of succinic acid in the treatment of autoimmune liver injury.

The effects of hyaluronan and proteoglycan link protein 1 (HAPLN1) in ameliorating spinal cord injury mediated by Nrf2.

Excessive inflammatory response and oxidative stress (OS) play an important role in the pathogenesis of spinal cord injury (SCI). Balance of inflammation and prevention of OS have been considered an effective strategy for the treatment of SCI. Hyaluronan and proteoglycan link protein 1 (HAPLN1), also known as cartilage link protein, has displayed a wide range of biological and physiological functions in different types of tissues and cells. However, whether HAPLN1 regulates inflammation and OS during SCI is unknown. Therefore, we aimed to examine whether HAPLN1 can have a protective effect on SCI. In this study, both in vitro and in vivo SCI models were established. Nissl staining and terminal deoxynucleotidyl transferase dUTP nick end labeling staining assays were used. Western blotting and enzyme-linked immunosorbent assay were employed to assess the expression of proteins. Our results demonstrate that the administration of HAPLN1 promoted the recovery of motor neurons after SCI by increasing the Basso mouse scale score, increasing the numbers of motor neurons, and preventing apoptosis of spinal cord cells. Additionally, HAPLN1 mitigated OS in spinal cord tissue after SCI by increasing the content of superoxide dismutase SOD and the activity of glutathione peroxidase but reducing the levels of malondialdehyde. Importantly, we found that HAPLN1 stimulated the activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway and stimulated the expression of heme oxygenase-1 and nicotinamide adenine dinucleotide phosphate quinone oxidoreductase-1, which mediated the attenuation of HAPLN1 in activation of the NOD-like receptor protein 3 (NLRP3) inflammasome by reducing the levels of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase-1, and interleukin-1β. Correspondingly, in vitro experiments show that the presence of HAPLN1 suppressed the NLRP3 inflammasome and prevented cell injury against H2O2 in PC12 cells. These effects were mediated by the Nrf2/ARE pathway, and inhibition of Nrf2 with ML385 abolished the beneficial effects of HAPLN1. Based on these findings, we conclude that HAPLN1 inhibits the NLRP3 inflammasome through the stimulation of the Nrf2/ARE pathway, thereby suppressing neuroinflammation, enhancing motor neuronal survival, and improving the recovery of nerve function after SCI.

The ACE2/Ang-(1-7)/MasR axis alleviates brain injury after cardiopulmonary resuscitation in rabbits by activating PI3K/Akt signaling.

Death among resuscitated patients is mainly caused by brain injury after cardiac arrest/cardiopulmonary resuscitation (CA/CPR). The angiotensin converting enzyme 2 (ACE2)/angiotensin (Ang)-(1-7)/Mas receptor (MasR) axis has beneficial effects on brain injury. Therefore, we examined the roles of the ACE2/Ang-(1-7)/MasR axis in brain injury after CA/CPR. We used a total of 76 male New Zealand rabbits, among which 10 rabbits underwent sham operation and 66 rabbits received CA/CPR. Neurological functions were determined by assessing serum levels of neuron-specific enolase and S100 calcium-binding protein B and neurological deficit scores. Brain water content was estimated. Neuronal apoptosis in the hippocampus was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling assays. The expression levels of various genes were measured by enzyme-linked immunosorbent assay and western blotting. Ang-(1-7) (MasR activator) alleviated CA/CPR-induced neurological deficits, brain edema, and neuronal damage, and A779 (MasR antagonist) had the opposite functions. The stimulation of ACE2/Ang-(1-7)/MasR inactivated the ACE/Ang II/AT1R axis and activated PI3K/Akt signaling. Inhibiting PI3K/Akt signaling inhibited Ang-(1-7)-mediated protection against brain damage after CA/CPR. Collectively, the ACE2/Ang-(1-7)/MasR axis alleviates CA/CPR-induced brain injury through attenuating hippocampal neuronal apoptosis by activating PI3K/Akt signaling.

Repurposing the diuretic benzamil as an anti-osteosarcoma agent that acts by suppressing integrin/FAK/STAT3 signalling and compromising mitochondrial function.

Osteosarcoma is the most common primary bone malignancy among children and adolescents. We investigated whether benzamil, an amiloride analogue and sodium-calcium exchange blocker, may exhibit therapeutic potential for osteosarcoma in vitro. MG63 and U2OS cells were treated with benzamil for 24 hours. Cell viability was evaluated with the MTS/PMS assay, colony formation assay, and flow cytometry (forward/side scatter). Chromosome condensation, the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, cleavage of poly-ADP ribose polymerase (PARP) and caspase-7, and FITC annexin V/PI double staining were monitored as indicators of apoptosis. Intracellular calcium was detected by flow cytometry with Fluo-4 AM. The phosphorylation and activation of focal adhesion kinase (FAK) and signal transducer and activator of transcription 3 (STAT3) were measured by western blot. The expression levels of X-linked inhibitor of apoptosis protein (XIAP), B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-extra large (Bcl-xL), SOD1, and SOD2 were also assessed by western blot. Mitochondrial status was assessed with tetramethylrhodamine, ethyl ester (TMRE), and intracellular adenosine triphosphate (ATP) was measured with BioTracker ATP-Red Live Cell Dye. Total cellular integrin levels were evaluated by western blot, and the expression of cell surface integrins was assessed using fluorescent-labelled antibodies and flow cytometry. Benzamil suppressed growth of osteosarcoma cells by inducing apoptosis. Benzamil reduced the expression of cell surface integrins α5, αV, and β1 in MG63 cells, while it only reduced the expression of αV in U2OS cells. Benzamil suppressed the phosphorylation and activation of FAK and STAT3. In addition, mitochondrial function and ATP production were compromised by benzamil. The levels of anti-apoptotic proteins XIAP, Bcl-2, and Bcl-xL were reduced by benzamil. Correspondingly, benzamil potentiated cisplatin- and methotrexate-induced apoptosis in osteosarcoma cells. Benzamil exerts anti-osteosarcoma activity by inducing apoptosis. In terms of mechanism, benzamil appears to inhibit integrin/FAK/STAT3 signalling, which triggers mitochondrial dysfunction and ATP depletion.

6-Gingerol attenuates hepatic ischemia/reperfusion injury through regulating MKP5-mediated P38/JNK pathway

6-Gingerol, the main bioactive compound of ginger, has antioxidant, anti-inflammatory, anti-cancer and neuroprotective effects. However, it is unclear whether 6-Gingerol has protective effects against hepatic ischemia/reperfusion (I/R) injury. In this study, the mouse liver I/R injury model and the mouse AML12 cell hypoxia/reoxygenation (H/R) model were established by pretreatment with 6-Gingerol at different concentrations to explore the potential effects of 6-Gingerol. Serum transaminase levels, liver necrotic area, cell viability, inflammatory response, and cell apoptosis were used to assess the effect of 6-Gingerol on hepatic I/R or cell H/R injury. Quantitative polymerase chain reaction (qPCR) and Western blotting were used to detect the mRNA and protein expression. The results show that 6-Gingerol decreased serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) levels, liver necrosis, inflammatory cytokines IL-1β, IL-6, MCP-1, TNF-α expression, Ly6g+ inflammatory cell infiltration, protein phosphorylation of NF-κB signaling pathway, Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) positive cells, cell apoptosis rate, the protein expression of pro-apoptotic protein BAX and C-Caspase3, increased cell viability, and expression of anti-apoptotic protein BCL-2. Moreover, 6-Gingerol could increase the mRNA and protein expression of mitogen activated protein kinase phosphatase 5 (MKP5) and inhibit the activation of P38/JNK signaling pathway. In MKP5 knockout (KO) mice, the protective effect of 6-gingerol and the inhibition of P38/JNK pathway were significantly weakened. Therefore, our results suggest that 6-Gingerol exerts anti-inflammatory and anti-apoptotic effects to attenuate hepatic I/R injury by regulating the MKP5-mediated P38/JNK signaling pathway.

Human Mesenchymal Stem Cells Improve Angiogenesis and Bone Formation in Severed Finger Rats through SIRT1/Nrf2 Signaling.

This study employed a severed finger rat model to analyze the effects of human mesenchymal stem cells (MSCs) on angiogenesis, inflammatory response, apoptosis, and oxidative stress, to evaluate the possible mechanism of the repair effect of MSCs on severed finger (SF) rats. Sixty Sprague-Dawley (SD) rats were categorized into five groups (n = 12). The pathological changes of severed finger tissues were investigated by Hematoxylin and eosin (H&E) staining on day 14 after the rats were sacrificed. The levels of inflammatory factors and oxidative stress factors were detected by ELISA. Terminal Deoxynucleotidyl Transferase (TdT) dUTP Nick End Labeling (TUNEL) was employed to assess the apoptosis of chondrocytes in severed finger tissues. The expression of osteocalcin (OCN), osteopontin (OPN), Collagen I (Col-1), and CD31 were detected by immunohistochemistry or immunofluorescence assay, respectively. The expression levels of related proteins were determined by western blot. Our study presented evidence that MSCs treatment improved pathological changes of skin and bone tissue, diminished the inflammatory response, prevented oxidative stress injury, suppressed chondrocyte apoptosis, and promoted angiogenesis, and bone formation compared to the model group. In addition, EX527 treatment attenuated the effect of MSCs, SRT1720 and ML385 co-treatment also attenuated the effect of MSCs. Importantly, the MSCs treatment increased the expression of Sirtuin 1(SIRT1)/Nuclear factor erythroid2-related factor 2(Nrf2) relate proteins. Our study indicated that the mechanism of the effect of MSCs on a severed finger was related to the SIRT1/ Nrf2 signaling pathway.