Expression Of Pathway Proteins Research Articles

Tirbanibulin decreases cell proliferation and downregulates protein expression of oncogenic pathways in human papillomavirus containing HeLa cells.

Tirbanibulin 1% ointment is a synthetic antiproliferative agent approved in 2021 by the European Union for treating actinic keratoses (AK). Topical tirbanibulin has clinically resolved HPV-57 ( +) squamous cell carcinoma (SCC), HPV-16 ( +) vulvar high-grade squamous intraepithelial lesion, epidermodysplasia verruciformis, and condyloma. We examined how tirbanibulin might affect HPV oncoprotein expression and affect other cellular pathways involved in cell proliferation and transformation. We treated the HeLa cell line, containing integrated HPV-18, with increasing doses of tirbanibulin to determine the effects on cell proliferation. Immunoblotting was performed with antibodies against theSrc canonical pathway, HPV 18 E6 and E7 transcription regulation, apoptosis, and invasion and metastasis pathways. Cell proliferation assays with tirbanibulin determined the half-maximal inhibitory concentration (IC50) of HeLa cells to be 31.49nmol/L. Increasing concentrations of tirbanibulin downregulates the protein expression of Src(p < 0.001), phospho-Src(p < 0.001), Ras(p < 0.01), c-Raf(p < 0.001), ERK1(p < 0.001), phospho-ERK1(p < 0.001), phospho-ERK2(p < 0.01), phospho-Mnk1(p < 0.001), eIF4E(p < 0.01), phospho-eIF4E(p < 0.001), E6(p < 0.01), E7(p < 0.01), Rb(p < 0.01), phospho-Rb(p < 0.001), MDM2(p < 0.01), E2F1(p < 0.001), phospho-FAK(p < 0.001), phospho-p130 Cas(p < 0.001), Mcl-1(p < 0.01), and Bcl-2(p < 0.001), but upregulates cPARP(p < 0.001), and cPARP/fPARP(p < 0.001). These results demonstrate that tirbanibulin may impact expression of HPV oncoproteins via the Src- MEK- pathway. Tirbanibulin significantly downregulates oncogenic proteins related to cell cycle regulation and cell proliferation while upregulating apoptosis pathways.

Modulatory Effects of Lactobacillus paracasei-Fermented Turmeric on Metabolic Dysregulation and Gut Microbiota in High-Fat Diet-Induced Obesity in Mice.

Turmeric, derived from Curcuma longa, and Lactobacillus paracasei, a lactic acid bacteria, have been studied for their potential antiobesity effects. To date, the antiobesity effects of turmeric fermented with L. paracasei have not been sufficiently investigated. This study was conducted via oral administration of 5% L. paracasei-fermented (FT) and unfermented turmeric (UT) in diet over 16 weeks using high-fat diet (HFD)-induced obese C57BL/6J mice. Results showed that the curcuminoid content of turmeric decreased following fermentation. Furthermore, FT significantly suppressed weight gain and liver and visceral adipose tissue weight and reduced plasma metabolic parameters in both the UT and FT experimental groups. The effects of FT were more noticeable than those of the unfermented form. Moreover, FT downregulated the expression of adipogenesis, lipogenesis, and inflammatory-related protein, but upregulated liver β-oxidation protein SIRT 1, PPARα, and PGC-1α in perigonadal adipose tissue. Additionally, FT ameliorated insulin resistance by activating insulin receptor pathway protein expressions in visceral adipose tissues. FT also modulated gut microbiota composition, particularly in two beneficial bacteria, Akkermansia muciniphila and Desulfovibrio, as well as two short-chain fatty acid-producing bacteria: Muribaculum intestinale and Deltaproteobacteria. Our findings indicate that the modulation effect of FT may be an important pathway for its antiobesity mechanisms.

Zhiqiao Gancao decoction regulated JAK2/STAT3/ macrophage M1 polarization to ameliorate intervertebral disc degeneration

BackgroundZhiqiao Gancao decoction (ZQGCD) was created by Professor Gong Zhengfeng, a renowned Chinese medicine expert. Clinical studies have shown its efficacy in alleviating pain and enhancing lumbar function in intervertebral disc degeneration (IDD) patients. However, the precise mechanism of ZQGCD in treating IDD remains unclear. MethodsThe active components of ZQGCD were identified using Liquid chromatography-tandem mass spectrometry (LC-MS/MS). A rat model of intervertebral disc degeneration was established, and rats in each group received ZQGCD for three weeks. Assessment parameters included hyperalgesia status, observation of intervertebral disc tissue degeneration and macrophage infiltration, and analysis of JAK2/STAT3 pathway protein expression in the intervertebral disc. Primary macrophage M1 polarization was induced using LPS, with cells treated using the JAK2 inhibitor (AZD1480) and ZQGCD to evaluate macrophage polarization, cellular supernatant inflammatory factors, and JAK2/STAT3 pathway expression. Macrophage supernatant served as a conditioned medium to observe its effects on the proliferation of nucleus pulposus cells (NPCs) and the expression of collagen II and MMP3 proteins. ResultsA total of 81 active components were identified in ZQGCD. Following ZQGCD treatment, infiltrating macrophages in intervertebral disc tissues of model rats decreased, the content of M1 macrophages decreased, while the content of M2 macrophages increased, the expression of proinflammatory factors and pain-inducing factors in serum decreased, and the expression of substance P in intervertebral disc tissue decreased. Consequently, the intervertebral disc degeneration and hyperalgesia of rats were improved. In vitro studies revealed that LPS induced M1 macrophage polarization. By inhibiting the JAK2/STAT3 pathway, both JAK2 inhibitors and ZQGCD effectively suppressed M1 polarization, resulting in decreased levels of IL-1β, IL-6, TNF-α, and various other inflammatory factors. Consequently, this inhibition led to a delay in the degeneration of NPCs. ConclusionThere is macrophage infiltration in the intervertebral disc tissue of IDD rats, and JAK2/STAT3 pathway is activated, macrophages are polarized to M1 type, resulting in inflammatory microenvironment, leading to intervertebral disc degeneration and hyperalgesia. ZQGCD exhibited a delaying effect on IDD and improved hyperalgesia by inhibiting the JAK2/STAT3/macrophage M1 polarization pathway.

Identification of 8 candidate microsatellite instability loci in colorectal cancer and validation of the ACVR2A mechanism in the tumor progression

This study probes the utility of biomarkers for microsatellite instability (MSI) detection and elucidates the molecular dynamics propelling colorectal cancer (CRC) progression. We synthesized a primer panel targeting 725 MSI loci, informed by The Cancer Genome Atlas (TCGA) and ancillary databases, to construct an amplicon library for next-generation sequencing (NGS). K-means clustering facilitated the distillation of 8 prime MSI loci, including activin A receptor type 2A (ACVR2A). Subsequently, we explored ACVR2A’s influence on CRC advancement through in vivo tumor experiments and hematoxylin–eosin (HE) staining. Transwell assays gauged ACVR2A’s role in CRC cell migration and invasion, while colony formation assays appraised cell proliferation. Western blotting illuminated the impact of ACVR2A suppression on CRC’s PI3K/AKT/mTOR pathway protein expressions under hypoxia. Additionally, ACVR2A’s influence on CRC-induced angiogenesis was quantified via angiogenesis assays. K-means clustering of NGS data pinpointed 32 MSI loci specific to tumor and DNA mismatch repair deficiency (dMMR) tissues. ACVR2A emerged as a pivotal biomarker, discerning MSI-H tissues with 90.97% sensitivity. A curated 8-loci set demonstrated 100% sensitivity and specificity for MSI-H detection in CRC. In vitro analyses corroborated ACVR2A’s critical role, revealing its suppression of CRC proliferation, migration, and invasion. Moreover, ACVR2A inhibition under CRC-induced hypoxia markedly escalated MMP3, CyclinA, CyclinD1, and HIF1α protein expressions, alongside angiogenesis, by triggering the PI3K/AKT/mTOR cascade. The 8-loci ensemble stands as the optimal marker for MSI-H identification in CRC. ACVR2A, a central element within this group, deters CRC progression, while its suppression amplifies PI3K/AKT/mTOR signaling and angiogenesis under hypoxic stress.

ET1 acts as a potential plasma biomarker and therapeutic target in deep venous thrombosis rat model.

Deep venous thrombosis (DVT) is the third leading cause of death in cardiovascular disease, following heart attacks and strokes. Early diagnosis and intervention are crucial for effective DVT therapy. We aim to investigate whether endothelin-1 (ET-1) could serve as an early diagnostic marker or a potential therapeutic target in a DVT rat model. CCK8 assay, invasion assay, and flow cytometry were used to detect the proliferation, migration and apoptosis of HUVECs, respectively. Elisa assay was used to detect ET-1 and coagulation factor VII in cell supernatant and rat?s plasma. Western blot was used to detect antioxidant signaling protein. Inferior vena cava stenosis was used to construct the DVT rat model. Lentivirus mediated overexpression of ET-1 in HUVECs impaired the cell proliferation and migration, increased cell apoptosis, inhibited the antioxidant signaling pathway proteins expression (e.g., NQO1, GCLC, Nrf-2), and upregulated coagulation factor VII. Furthermore, overexpression of ET-1 further impaired antioxidant signaling pathway protein in response to H2O2 treatment. However, lentivirus mediated ET-1 knockdown and BQ123 (an ET-1 inhibitor), showed the opposite results with ET-1 overexpression. We then established a DVT rat model by inferior vena cava stenosis. The stenosis induced early expression of ET-1 and coagulation factor VII in plasma at day 1 and restore their level at day 10. BQ123 could downregulate the coagulation factor VII to ameliorate the stenosis effects. Our findings suggest that ET-1 might serve as an early diagnostic marker for DVT rat model and a potential therapeutic target for treating DVT.

Methylation-regulated tumor suppressor gene PDE7B promotes HCC invasion and metastasis through the PI3K/AKT signaling pathway.

Hepatocellular carcinoma (HCC) has a high mortality rate, and the mechanisms underlying tumor development and progression remain unclear. However, inactivated tumor suppressor genes might play key roles. DNA methylation is a critical regulatory mechanism for inactivating tumor suppressor genes in HCC. Therefore, this study investigated methylation-related tumor suppressors in HCC to identify potential biomarkers and therapeutic targets. We assessed genome-wide DNA methylation in HCC using whole genome bisulfite sequencing (WGBS) and RNA sequencing, respectively, and identified the differential expression of methylation-related genes, and finally screened phosphodiesterase 7B (PDE7B) for the study. The correlation between PDE7B expression and clinical features was then assessed. We then analyzed the changes of PDE7B expression in HCC cells before and after DNA methyltransferase inhibitor treatment by MassArray nucleic acid mass spectrometry. Furthermore, HCC cell lines overexpressing PDE7B were constructed to investigate its effect on HCC cell function. Finally, GO and KEGG were applied for the enrichment analysis of PDE7B-related pathways, and their effects on the expression of pathway proteins and EMT-related factors in HCC cells were preliminarily explored. HCC exhibited a genome-wide hypomethylation pattern. We screened 713 hypomethylated and 362 hypermethylated mCG regions in HCC and adjacent normal tissues. GO analysis showed that the main molecular functions of hypermethylation and hypomethylation were "DNA-binding transcriptional activator activity" and "structural component of ribosomes", respectively, whereas KEGG analysis showed that they were enriched in "bile secretion" and "Ras-associated protein-1 (Rap1) signaling pathway", respectively. PDE7B expression was significantly down-regulated in HCC tissues, and this low expression was negatively correlated with recurrence and prognosis of HCC. In addition, DNA methylation regulates PDE7B expression in HCC. On the contrary, overexpression of PDE7B inhibited tumor proliferation and metastasis in vitro. In addition, PDE7B-related genes were mainly enriched in the PI3K/ATK signaling pathway, and PDE7B overexpression inhibited the progression of PI3K/ATK signaling pathway-related proteins and EMT. PDE7B expression in HCC may be regulated by promoter methylation. PDE7B can regulate the EMT process in HCC cells through the PI3K/AKT pathway, which in turn affects HCC metastasis and invasion.

Revealing the potential bioactive components and mechanism of Qianhua Gout Capsules in the treatment of gouty arthritis through network pharmacology, molecular docking and pharmacodynamic study strategies

Recent clinical studies have confirmed the effectiveness of Qianhua Gout Capsules (QGC) in the treatment of gouty arthritis (GA). However, the specific regulatory targets and mechanisms of action of QGC are still unclear. To address this gap, we utilized network pharmacology, molecular docking, and pharmacodynamic approaches to investigate the bioactive components and associated mechanisms of QGC in the treatment of GA. By employing UPLC-Q Exactive-MS, we identified the compounds present in QGC, with active ingredients defined as those with oral bioavailability ≥30 % and drug similarity ≥0.18. Subsequently, the targets of these active compounds were determined using the TCMSP database, while GA-related targets were identified from DisGeNET, GeneCards, TTD, OMIM, and DrugBank databases. Further analysis including PPI analysis, GO analysis, and KEGG pathway enrichment was conducted on the targets. Validation of the predicted results was performed using a GA rat model, evaluating pathological changes, inflammatory markers, and pathway protein expression. Our results revealed a total of 130 components, 44 active components, 16 potential shared targets, GO-enriched terms, and 47 signaling pathways related to disease targets. Key active ingredients included quercetin, kaempferol, β-sitosterol, luteolin, and wogonin. The PPI analysis highlighted five targets (PPARG, IL-6, MMP-9, IL-1β, CXCL-8) with the highest connectivity, predominantly enriched in the IL-17 signaling pathway. Molecular docking experiments demonstrated strong binding of CXCL8, IL-1β, IL-6, MMP9, and PPARG targets with the top five active compounds. Furthermore, animal experiments confirmed the efficacy of QGC in treating GA in rats, showing reductions in TNF-α, IL-6, and MDA levels, and increases in SOD levels in serum. In synovial tissues, QGC treatment upregulated CXCL8 and PPARG expression, while downregulating IL-1β, MMP9, and IL-6 expression. In conclusion, this study applied a network pharmacology approach to uncover the composition of QGC, predict its pharmacological interactions, and demonstrate its in vivo efficacy, providing insights into the anti-GA mechanisms of QGC. These findings pave the way for future investigations into the therapeutic mechanisms underlying QGC's effectiveness in the treatment of GA.

TNF-α can promote membrane invasion by activating the MAPK/MMP9 signaling pathway through autocrine in bone-invasive pituitary adenoma.

A bone-invasive pituitary adenoma exhibits aggressive behavior, leading to a worse prognosis. We have found that TNF-α promotes bone invasion by facilitating the differentiation of osteoclasts, however, before bone-invasive pituitary adenoma invades bone tissue, it needs to penetrate the dura mater, and this mechanism is not yet clear. We performed transcriptome microarrays on specimens of bone-invasive pituitary adenomas (BIPAs) and noninvasive pituitary adenomas (NIPAs) and conducted differential expressed gene analysis and enrichment analysis. We altered the expression of TNF-α through plasmids, then validated the effects of TNF-α on GH3 cells and verified the efficacy of the TNF-α inhibitor SPD304. Finally, the effects of TNF-α were validated in invivo experiments. Pathway act work showed that the MAPK pathway was significantly implicated in the pathway network. The expression of TNF-α, MMP9, and p-p38 is higher in BIPAs than in NIPAs. Overexpression of TNF-α elevated the expression of MAPK pathway proteins and MMP9 in GH3 cells, as well as promoted proliferation, migration, and invasion of GH3 cells. Flow cytometry indicated that TNF-α overexpression increased the G2 phase ratio in GH3 cells and inhibited apoptosis. The expression of MMP9 was reduced after blocking the P38 MAPK pathway; overexpression of MMP9 promoted invasion of GH3 cells. Invivo experiments confirm that the TNF-α overexpression group has larger tumor volumes. SPD304 was able to suppress the effects caused by TNF-α overexpression. Bone-invasive pituitary adenoma secretes higher levels of TNF-α, which then acts on itself in an autocrine manner, activating the MAPK pathway and promoting the expression of MMP9, thereby accelerating the membrane invasion process. SPD304 significantly inhibits the effect of TNF-α and may be applied in the clinical treatment of bone-invasive pituitary adenoma.

Overexpression of TRIM44 mediates the NF-κB pathway to promote the progression of ovarian cancer.

Ovarian cancer (OC) is the second most commonly seen cancer in the US, and patients with OC are commonly diagnosed in the advanced stage. Research into the molecular mechanisms and potential therapeutic targets of OC is becoming increasingly urgent. In our study, we worked to discover the role of TRIM44 in OC development. This study explored whether the overexpression of TRIM44 mediates the NF-kB pathway to promote the progression of OC. A TRIM44 overexpression model was constructed in SKOV3 cells, and the proliferation ability of the cells was detected using the CCK-8 assay. The migration healing ability of cells was detected using cell scratch assay. Cell migration and invasion were detected using Transwell nesting. TUNEL was applied to detect apoptosis, and ELISA and western blot were used to detect the expression of NF-κB signaling pathway proteins. The pathological changes of the tumor tissues were observed using HE staining in a mouse ovarian cancer xenograft model. Immunofluorescence double staining, RT-PCR, and western blot were used to determine the expression of relevant factors in tumour tissues. TRIM44 overexpression promoted the proliferation, migration, and invasion of SKOV3 cells in vitro and inhibited apoptosis while enhancing the growth of tumours in vivo. TRIM44 regulated the NF-κB signaling pathway. TRIM44 overexpression can regulate the NF-κB signaling pathway to promote the progression of OC, and TRIM44 may be a potential therapeutic target for OC.

Regulation of optimized new Shengmai powder on cardiomyocyte apoptosis and ferroptosis in ischemic heart failure rats: The mediating role of phosphatidylinositol-3-kinase/protein kinase B/tumor protein 53 signaling pathway

Ethnopharmacological relevanceOptimized New Shengmai Powder (ONSMP) is a sophisticated traditional Chinese medicinal formula renowned for bolstering vital energy, optimizing blood circulation, and mitigating fluid retention. After years of clinical application, ONSMP has shown a significant impact in improving myocardial injury and cardiac function and has a positive effect on treating heart failure. However, many unknowns exist about the molecular biological mechanisms of how ONSMP exerts its therapeutic effects, which require further research and exploration. Aim of the studyExploring the potential molecular biological mechanisms by which ONSMP ameliorates cardiomyocyte apoptosis and ferroptosis in ischemic heart failure (IHF). Materials and methodsFirst, we constructed a rat model of IHF by inducing acute myocardial infarction through surgery and using echocardiography, organ coefficients, markers of heart failure, antioxidant markers, and histopathological examination to assess the effects of ONSMP on cardiomyocyte apoptosis and ferroptosis in IHF rats. Next, we used bioinformatics analysis techniques to analyze the active components, signaling pathways, and core targets of ONSMP and calculated the interactions between core targets and corresponding elements. Finally, we detected the positive expression of apoptosis and ferroptosis markers and core indicators of signaling pathways by immunohistochemistry; detected the mean fluorescence intensity of core indicators of signaling pathways by immunofluorescence; detected the protein expression of signaling pathways and downstream effector molecules by western blotting; and detected the mRNA levels of p53 and downstream effector molecules by quantitative polymerase chain reaction. ResultsONSMP can activate the Ser83 site of ASK by promoting the phosphorylation of the PI3K/AKT axis, thereby inhibiting the MKK3/6-p38 axis and the MKK4/7-JNK axis signaling to reduce p53 expression, and can also directly target and inhibit the activity of p53, ultimately inhibiting p53-mediated mRNA and protein increases in PUMA, SAT1, PIG3, and TFR1, as well as mRNA and protein decreases in SLC7A11, thereby inhibiting cardiomyocyte apoptosis and ferroptosis, effectively improving cardiac function and ventricular remodeling in IHF rat models. ConclusionONSMP can inhibit cardiomyocyte apoptosis and ferroptosis through the PI3K/AKT/p53 signaling pathway, delaying the development of IHF.

Osthole ameliorates early diabetic kidney damage by suppressing oxidative stress, inflammation and inhibiting TGF-β1/Smads signaling pathway

BackgroundOsthole is a natural active ingredient extracted from the traditional Chinese medicine Cnidium monnieri. It has been demonstrated to have anti-inflammatory, anti-fibrotic, and anti-hyperglycemic properties. However, its effect on diabetic kidney disease (DKD) remains uncertain. This study aims to assess the preventive and therapeutic effects of osthole on DKD and investigate its underlying mechanisms. MethodsA streptozotocin/high-fat and high-sucrose diet induced Type 2 diabetic rat model was established. Metformin served as the positive drug control. Diabetic rats were treated with metformin or three different doses of osthole for 8 weeks. Throughout the treatment period, the progression of DKD was assessed by monitoring increases in urinary protein, serum creatinine, urea nitrogen, and uric acid, along with scrutinizing kidney pathology. Enzyme-linked immunosorbent assay (ELISA) was employed to detect inflammatory factors and oxidative stress levels. At the same time, immunohistochemical staining was utilized to evaluate changes in alpha-smooth muscle actin, fibronectin, E-cadherin, and apoptosis. The alterations in TGF-β1/Smads signaling pathway were ascertained through western blot and immunofluorescence. Furthermore, we constructed a high glucose-stimulated HBZY-1 cells model to uncover its molecular protective mechanism. ResultsOsthole significantly reduced fasting blood glucose, insulin resistance, serum creatinine, uric acid, blood urea nitrogen, urinary protein excretion, and glomerular mesangial matrix deposition in diabetic rats. Additionally, significant improvements were observed in inflammation, oxidative stress, apoptosis, and fibrosis levels. The increase of ROS, apoptosis and hypertrophy in HBZY-1 cells induced by high glucose was reduced by osthole. Immunofluorescence and western blot results demonstrated that osthole down-regulated the TGF-β1/Smads signaling pathway and related protein expression. ConclusionOur findings indicate that osthole exhibits potential preventive and therapeutic effects on DKD. It deserves further investigation as a promising drug for preventing and treating DKD.

Acrylamide-Aggravated Liver Injury by Activating Endoplasmic Reticulum Stress in Female Mice with Diabetes.

Acrylamide (ACR) is a common industrial contaminant with endocrine-disrupting toxicity. Numerous studies have indicated that females and diabetics are more sensitive to environmental contaminants. However, it remains unknown whether female diabetics are susceptible to ACR-induced toxicity and its potential mechanisms. Thus, the female ACR-exposure diabetic Balb/c mice model was established to address these issues. Results showed that ACR could induce liver injury in normal mice and cause more serious inflammatory cell infiltration, hepatocyte volume increase, and fusion in diabetic mice liver. Meanwhile, ACR could lead to exacerbation of diabetic symptoms in diabetic mice by disturbing the glucose and lipid metabolism in the liver, which mainly manifests as the accumulation of liver glycogen and liver lipids, the reduction of the activity/content of glycolytic and metabolizing enzyme as well as pentose phosphatase, upregulation of the gene expression in fatty acid transporter and gluconeogenesis, and downregulation of the gene expression in fatty acid synthesis and metabolism. Moreover, ACR exposure could induce oxidative stress, inflammation, and endoplasmic reticulum stress in the liver by a decrease in hepatic antioxidant enzyme activity and antioxidant content, an increase in inflammatory factor levels, and a change in the related protein expression of endoplasmic reticulum stress (ERS) and apoptosis-related pathways in diabetic mice. Statistical analysis results revealed that ACR-induced liver injury was highly correlated with inflammation and oxidative stress, and ERS and diabetic mice had a higher risk of liver injury than normal mice. Overall results suggested that female diabetic mice easily suffer from ACR-induced toxicity, and the reason was that ACR could induce further damage to the liver by worsening the condition of inflammation, oxidative stress, and ERS in the liver.

Comparative study of leucine on digestive enzyme secretion, ISR and mTOR pathway in cattle and porcine pancreas in vitro

ABSTRACT This experiment is to investigate whether interspecies differences exist in the effects of leucine (Leu) on digestive enzyme secretion in pancreatic tissues through in vitro. Five healthy Simmental cattle and Duroc × Landrace × Yorkshire growing-finishing pigs were selected, and pancreatic tissues were taken for culture after slaughter. Leu levels of 0, 2.62, 5.24, 10.48 mg/mL. Leu levels of 0, 5.24 mg/mL were selected to determine the expression of mRNA and proteins in the signaling pathways. The results showed that Leu increased the activity of digestive enzyme in cattle and pig, and there was an interaction was found on trypsin and chymotrypsin (P < 0.05). Leu had a significant effect on the mRNA (PKB, S6K1, 4E-BP1 and ATF4) and protein expression (PKB and 4E-BP1) and the phosphorylation (PKB, S6K1 and ATF4) levels (P < 0.05). Animal species had significant effects on the mRNA (PKB, S6K1 and ATF4) and protein (S6K1 and 4E-BP1) expression, and the phosphorylation (4E-BP1) levels (P < 0.05). There was an interaction between animal species and Leu on the expression of protein (PKB and S6K1) (P < 0.05). In summary, interspecies differences exist in the regulation of Leu on digestive enzyme secretion in pancreatic tissues in vitro of different animals.

LINC00942 Alleviates NaAsO2-induced Apoptosis by Promoting GSH Synthesis Through Targeting miR-214-5p.

The mechanism of arsenic-induced liver toxicity is not fully understood. This study aimed to investigate the role of LINC00942 in arsenic-induced hepatotoxicity by regulating miR-214-5p. As the exposure dose of NaAsO2 gradually increases, cell viability, intracellular GSH content, ΔΨm, and the protein levels of GCLC and GCLM were reduced significantly. Apoptosis rate, ROS, and expression of apoptosis-related and NF-κB pathway proteins increased. The expression of LINC00942 was increased, while the expression of miR-214-5p was decreased. After suppressing LINC00942 levels, NaAsO2 exposure further decreased cell viability, intracellular GSH content, ΔΨm, GCLC protein, and miR-214-5p expression. The apoptosis rate, ROS, and apoptosis-related and NF-κB pathway proteins further increased. miR-214-5p is targeted and negatively regulated by LINC00942. After miR-214-5p was overexpressed, NaAsO2 further decreased cell viability, intracellular GSH content, ΔΨm, and GCLC protein expression compared to NaAsO2 exposure. The apoptosis rate, ROS, apoptosis-related and NF-κB pathway proteins p65, and IKKβ were higher than those exposed to NaAsO2. LINC00942 inhibitor along with miR-214-5p inhibitor combined with NaAsO2 treatment resulted in increased cell viability, GSH, Bcl-2, and GCLC protein expression and decreased apoptosis rate, apoptosis related, p65, IKKβ protein, and ΔΨm, as compared to the combined NaAsO2 and si LINC00942 group. NaAsO2 exposure induces oxidative damage and apoptosis in LX-2 cells by activating NF-κB and inhibiting GSH synthesis. During this process, the expression level of LINC00942 increases, targeting to reduce the level of miR-214-5p, then weakening the effect of NaAsO2 on NF-κB, thereby alleviating cellular oxidative damage and playing a protective role.

Paeonol attenuates nonalcoholic steatohepatitis by regulating intestinal flora and AhR/NLRP3/Caspase-1 metabolic pathway

Ethnopharmacological relevanceNon-alcoholic steatohepatitis (NASH) is a common metabolic liver injury disease that is closely associated with obesity and metabolic disorders. Paeonol, an active ingredient found in Moutan Cortex, a traditional Chinese medicine which exhibits significant therapeutic effect on liver protection, has shown promising effects in treating liver diseases, particularly NASH. However, the specific intervention mechanism of paeonol on NASH is still unknown. Aim of the studyOur objective is to elucidate the pharmacological mechanism of paeonol in intervening NASH at the in vivo level, focusing on the impact on intestinal flora, tryptophan-related targeted metabolome, and related Aryl hydrocarbon receptor (AhR) pathways. Materials and methodsHere, we explored the intervention effect of paeonol on NASH by utilizing the NASH mouse model. The Illumina highthroughput sequencing technology was preformed to determine the differences of gut microbiota of model and paeonol treatment group. The concentration of Indoleacetic acid is determined by ELISA. The intervention effect of NASH mouse and AhR/NLRP3/Caspase-1 metabolic pathway is analyzed by HE staining, oil red O staining, Immunohistochemistry, Immunofluorescence, Western blot and qRT-PCR assays. Fecal microbiota transplantation experiment also was performed to verify the intervention effect of paeonol on NASH by affecting gut microbiota. ResultsFirstly, we discovered that paeonol effectively reduced liver pathology and blood lipid levels in NASH mice, thereby intervening in the progression of NASH. Subsequently, through 16S meta-analysis, we identified that paeonol can effectively regulate the composition of intestinal flora in NASH mice, transforming it to resemble that of normal mice. Specifically, paeonol decreased the abundance of certain Gram-negative tryptophan-metabolizing bacteria. Moreover, we discovered that paeonol significantly increased the levels of metabolites Indoleacetic acid, subsequently enhancing the expression of AhR-related pathway proteins. This led to the inhibition of the NOD-like receptor protein 3 (NLRP3) inflammasome production and inflammation generation in NASH. Lastly, we verified the efficacy of paeonol in intervening NASH by conducting fecal microbiota transplantation experiments, which confirmed its role in promoting the AhR/NLRP3/cysteinyl aspartate specific proteinase (Caspase-1) pathway. ConclusionsOur findings suggest that paeonol can increase the production of Indoleacetic acid by regulating the gut flora, and promote the AhR/NLRP3/Caspase-1 metabolic pathway to intervene NASH.

Bio-inspired deposition of gold nanoparticles onto the surface of kaolin for in vitro management of human ovarian cancer and modulation of the inflammatory response in adenomyosis-induced mice in vivo via the MAPK signaling pathway

A mild and eco-friendly protocol has been developed for the preparation of kaolin-decorated Au nanoparticles mediated by Ephedra root extract as a green reducing and stabilizing agents without any toxic substrates. Structural features of the prepared Au NPs/Kaolin were assessed through FE-SEM, TEM, and XRD techniques. TEM images show the good deposition of Au NPs over the surface of extract-modified kaolin without aggregation. Towards the medicinal application, its antioxidant efficacy was assessed by the DPPH method, and the corresponding IC50 value was obtained as 104 μg/mL. Cytotoxicity of the nanoformulated bio-composite was ascertained through MTT analysis against human ovarian carcinoma cells, i.e., PA-1 and SK-OV-3. The IC50 in those studies was 250 and 119 μg/mL against PA-1and SK-OV-3 cells, respectively. In the in vivo design, tamoxifen was used to induce the experimental adenomyosis model in mice. After treatment, the thymus, spleen, uterine, and body weights of all animals were measured. Then, inflammatory factor expression and myometrial infiltration were determined by qRT-PCR, ELISA, and histology examination in the uterus. Western blotting, qRT-PCR, and immune histochemical (IHC) staining were applied to analyze the MAPK/ERK signaling pathway protein expression. Au NPs/Kaolin bio-nanocomposite ameliorated the adenomyosis symptoms by raising the thymus and spleen index and decreasing the myometrial infiltration. The raised levels of TNF-α, IL-6, and IL-1β in adenomyosis model mice uterus and serum were also reduced after Au NPs/Kaolin bio-nanocomposite treatment. The adenomyosis amelioration of Au NPs/Kaolin bio-nanocomposite was gained by preventing the MAPK/ERK signaling pathway, including decreasing the expressions of protein and mRNA of p-p38/p38, p-JNK/JNK, and p-ERK/ERK.

Knockdown of RGMA improves ischemic stroke via Reprogramming of Neuronal Metabolism

Neuronal energy metabolism dysregulation is involved in various pathologies of Ischemia-reperfusion (I/R), yet the role of RGMA in neuronal metabolic reprogramming has not been reported. In this study, we found that RGMA expression significantly increased after I/R, and compared to control mice, mice with MCAO/R showed an increase in glycolytic metabolic products and the expression of glycolytic pathway proteins. Furthermore, RGMA levels are closely related to neuronal energy metabolism. We discovered that knockdown of RGMA can shift neuronal energy metabolism towards oxidative phosphorylation and the pentose phosphate pathway, thereby protecting mice from ischemic reperfusion injury. Mechanistically, knockdown of RGMA can downregulate PGK1 expression, reducing the increase in glycolytic flux following ischemia reperfusion. Moreover, we found that knockdown of RGMA can reduce the interaction between USP10 and PGK1, thus affecting the ubiquitination degradation of PGK1. In summary, our data suggest that RGMA may regulate neuronal energy metabolism by inhibiting the USP10-mediated deubiquitination of PGK1, thus protecting it from I/R injury. This study provides new ideas for clarifying the intrinsic mechanism of neuronal damage after I/R.

Enhancing Fat Transplantation Efficiency in a Mouse Model through Pretreatment of Adipose-Derived Stem Cells with RIP3 Inhibitors.

Autologous fat transplantation, widely used in cosmetic and reparative surgery for volumetric enhancements, faces challenges with its inconsistent long-term survival rates. The technique's efficacy, crucial for its development, is hindered by unpredictable outcomes. Enriching fat grafts with adipose-derived stem cells (ADSCs) shows promise in improving survival efficiency. This study aimed to explore the potential of receptor-interacting protein kinase 3 (RIP3) kinase inhibitors as a pretreatment for ADSCs in enhancing autologous fat graft retention over a long term. ADSCs were isolated, cultured under normal or oxygen-glucose deprivation conditions, and mixed with particulate fat grafts to form distinct experimental groups in female nude mice. Fat graft mass and volume, along with underlying mechanisms, were evaluated using quantitative reverse transcription polymerase chain reaction (RT-qPCR), immunohistochemistry, and Western blot analysis. The experimental group, pretreated with RIP3 kinase inhibitors, had higher graft mass and volume, greater adipocyte integrity, and increased peroxisome proliferator-activated receptor gamma (PPARγ) mRNA levels than control groups. Furthermore, the experimental group demonstrated lower expression of necroptosis pathway proteins in the short term and an ameliorated inflammatory response as indicated by interleukin-1 beta (IL-1β), interleukin-10 (IL-10) mRNA levels, and histological analyses. Notably, enhanced neovascularization was evident in the experimental group. These findings suggest that RIP3 kinase inhibitor pretreatment of ADSCs can improve fat graft survival, promote adipocyte integrity, potentially decrease inflammation, and enhance neovascularization. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Abstract 7219: Preclinical investigations of concomitant tumor treating fields (TTFields) with cisplatin for treatment of cervical cancer

Abstract Introduction: Cervical cancer, although largely preventable, continues to be a significant health concern. Early-stage treatment is commonly surgery; while advanced, recurrent, or metastatic disease often requires systemic therapy. Chemotherapy, mainly cisplatin is the backbone of first-line therapy. TTFields are electric fields that disrupt cellular processes critical for cancer cell viability and tumor progression. In other tumor types, TTFields applied with cisplatin has exhibited benefit. This in vitro study assessed the preclinical efficacy of TTFields for cervical cancer and explored efficacy for concurrent use with cisplatin. Methods: Effects on cell count in cervical cancer cell lines exposed to TTFields (0.5 V/cm for Ca Ski cells, 1 V/cm RMS for HeLa and SiHa cells; 72 h) across a range of frequencies (100-400 kHz) were evaluated. TTFields (200 kHz) were then applied with varying cisplatin doses followed by cell count, colony formation, and apoptosis assays. Overall effect was calculated as the product of percent reductions in cell count and colony formation. TTFields-treated cells were evaluated for formation of the DNA damage marker γH2AX using fluorescent microscopy; and for the expression of Fanconi Anemia (FA)-BRCA pathway proteins through Western blotting. Results: TTFields treatment reduced cell count in all cervical cancer cell lines tested. Cisplatin exhibited dose-response effects, which were enhanced when TTFields were co-applied. TTFields led to downregulated expression of FA-BRCA pathway proteins, which are involved in repairing cisplatin-induced DNA damage, resulting in increased DNA damage. Conclusions: These preclinical results suggest that TTFields should be further explored as a potential treatment for cervical cancer. TTFields-induced downregulation of the FA-BRCA pathway possibly explains the enhanced efficacy when TTFields are applied together with cisplatin, providing mechanistic insight. Citation Format: Roni Frechtel-Gerzi, Daria Gerasimova, Einav Zeevi, Roni Monin, Hila Fishman, Inbar Schlachet-Drukerman, Helena Mumblat, Antonia Martinez-Conde, Rotem Engelman, Eyal Dor-On, Itai Tzchori, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti, Greg M. Palmer, Angeles Alvarez Secord. Preclinical investigations of concomitant tumor treating fields (TTFields) with cisplatin for treatment of cervical cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7219.

Abstract 5929: HuR-targeted therapy promotes the adaptive immune response in medulloblastoma through the cGAS-Sting pathway

Abstract HuR is an RNA binding protein overexpressed in multiple human cancers and is involved in tumorigenesis, invasion, and metastasis. Genetic and pharmacologic inhibition of HuR demonstrated antitumor activity establishing HuR as a molecular target for cancer therapy. However, little is known about HuR’s role in medulloblastoma (MB). In the present study, we investigated the effect of CMLD2, a small molecule inhibitor of HuR, on MB cell growth and on the adaptive immune response by assessing the cyclic GMP-AMP synthase (cGAS) - stimulator of interferon genes (STING) pathway. The cytoplasmic double-stranded DNA (dsDNA) sensing cGAS-STING signaling pathway plays pivotal role in host defense against cancers. Activation of the cGAS-STING signaling cascade induces the expression of pro-inflammatory cytokines and type I interferons promoting anti-tumor immunity. Human MB Daoy cells were treated with DMSO (control) or CMLD2 (20uM and 30uM). At 48 and 72 h after treatment, cells were collected and cell viability was determined using Trypan blue assay. A time and dose-dependent cytotoxicity was observed with 22.5% and 32.5% reduction in cell number with 20uM treatment (p&amp;lt;0.01) and 38.3% and 50.2% reduction with 30uM treatment (p&amp;lt;0.001) at 48 h and 72 h respectively compared to DMSO-treated control. Western blot analysis showed CMLD2 treatment inhibited HuR and markedly increased the expression of cGAS-STING pathway proteins (p-STING Ser366, p-TBK1 Ser172, p-IRF3Ser396 and pNF-KBSer536) at the two drug concentrations tested albeit greater increase in cGAS-STING protein expression was observed with 20uM treatment. Analysis for chemokines (CXCL10 and CCL5) and cytokine (IL-6) associated with the cGAS-STING pathway in cell culture supernatant collected from 20uM CMLD2 treatment showed significant increase in CXCL10 (1.96 ± 0.12 and 2.12 ± 0.06 fold increase at 48 h and 72 h; p&amp;lt;0.01) and CCL5 (1.21 ± 0.09 and 1.86 ± 0.05 fold increase at 48 h and 72 h; p=ns at 48 h; p&amp;lt;0.01 at 72 h) over DMSO-treated control. IL-6 expression significantly increased at 72h (1.98± 0.24 fold, p&amp;lt;0.001) but not at 48h after 20uM CMLD2 treatment compared to DMSO treated control. Our study results demonstrate CMLD2 mediated HuR targeting induces MB cell cytotoxicity and concurrently activates the cGAS-STING pathway. Testing of CMLD2 treatment in additional MB cell models and studying the impact of the cGAS-STING activation on immune cells (T cells, NK cells and macrophages) will aid in developing novel HuR-targeted treatment strategies in MB. Funding: The study was supported in part by a grant received from the National Cancer Institute of the National Institutes of Health (R01 CA 282735-01) and funds from the Jim and Christy Everest Endowed Chair in Cancer Developmental Therapeutics. Citation Format: Meghna Mehta, Rajeswari Raguraman, Anupama Munshi, Rajagopal Ramesh. HuR-targeted therapy promotes the adaptive immune response in medulloblastoma through the cGAS-Sting pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5929.