Expression Of Related Molecules Research Articles

Cinobufotalin inhibits proliferation, migration and invasion in hepatocellular carcinoma by triggering NOX4/NLRP3/GSDMD-dependent pyroptosis

IntroductionPyroptosis is an inflammatory form of programmed cell death that plays a significant role in tumorigenesis. Cinobufotalin (CB), a bufadienolide extracted from toad venom, is associated with antitumor effects in various cancers, including liver cancer. However, the role of CB in pyroptosis and its underlying mechanisms have not been well characterized.MethodsMTT, Colony formation, EdU, Wound healing and Transwell migration and invasion assays were applied to determine the effects of CB on the proliferation, migration, and invasion ability of hepatocellular carcinoma (HCC) cells in vitro. The subcutaneous xenograft mouse model and pulmonary metastasis model were used to evaluate the effect of CB on HCC cells in vivo. PCR, western blot, immunohistochemistry, immunofluorescence, and ELISA were used to verify the expression of proliferation, migration, pyroptosis, and inflammation related molecules after CB treatment. Using si-RNA and inhibitors to interfere with NOX4 and HLRP3 expression to validate the key signaling pathways of pyroptosis induced by CB treatment.ResultsIn vivo experiments using nude mice with xenografted HCC cells and in vitro experiments with HCC cell lines demonstrated that CB treatment significantly inhibited the proliferation, migration, and invasiveness of HCC cells. CB treatment also showed dose-dependent activation of the NLRP3 inflammasome complex in HCC cells, leading to gasdermin D-induced pyroptosis. However, these effects were abrogated via the pretreatment of HCC cells with VX-765, a caspase-1 inhibitor. Additionally, CB increased the production of reactive oxygen species (ROS) and H₂O₂, along with upregulating NOX4 protein expression in HCC cells. Conversely, NOX4 silencing or pretreatment with VAS2870 (an NOX4 inhibitor) or NAC (an ROS scavenger) suppressed the activation of the NLRP3 inflammasome complex and pyroptosis in CB-treated HCC cells.DiscussionOur study demonstrated that CB suppressed the proliferation, migration, and invasiveness of HCC cells by inducing pyroptosis through the activation of the NOX4/NLRP3/GSDMD signaling pathway. Therefore, our results suggest that CB is a promising therapeutic agent for HCC.

The expression and role of SUZ12 in lung adenocarcinoma.

SUZ12 is one of the core members of the polycomb repressive complex 2 (PRC2), but its expression and role in lung adenocarcinoma (LUAD) are unclear. We aimed to explore the expression, prognosis, biological functions and roles of SUZ12 in LUAD. The expression of SUZ12 was detected by immunohistochemical staining, qRT-PCR, and western blotting in LUAD tissues and cells. The biological functions and molecular mechanisms of SUZ12 were characterized by a range of invitro and invivo experiments. SUZ12 was overexpressed in LUAD tissues, and high SUZ12 expression was correlated with worse clinicopathological features and a poorer prognosis. Knockdown of SUZ12 significantly inhibited cell growth, colony formation, invasion, and migration, and induced apoptosis and G1/S phase arrest, while overexpression of SUZ12 had the opposite effects. Knockdown of SUZ12 decreased the tumorigenic capacity of A549 cells invivo. The expression of key signaling molecules related to the cell cycle, apoptosis, migration, and immunity were altered by the knockdown or overexpression of SUZ12. SUZ12 can directly bind to the Bax promoter region, EZH2 and H3K27me3 levels dependents on SUZ12. The expression levels of SUZ12 and Bax were negatively correlated in LUAD tissues. SUZ12 is a new oncogene related to the poor prognosis of LUAD. SUZ12 regulates LUAD progression by regulating the expression of related signaling molecules, and as a part of the PRC2 complex, it may bind to the Bax promoter to silence Bax expression.

Gastrodin exerts perioperative myocardial protection by improving mitophagy through the PINK1/Parkin pathway to reduce myocardial ischemia-reperfusion injury

BackgroundAlthough blood flow is restored after treatment of myocardial infarction (MI), myocardial ischemia and reperfusion (I/R) can cause cardiac injury, which is a leading cause of heart failure. Gastrodin (GAS) exerts protective effects against brain, heart, and kidney I/R. However, its pharmacological mechanism in myocardial I/R injury (MIRI) remains unclear. PurposeGAS regulates autophagy in various diseases, such as acute hepatitis, vascular dementia, and stroke. We hypothesized that GAS could repair mitochondrial damage and regulate autophagy to protect against MIRI. Study designMale C57BL/6 mice and H9C2 cells were subjected to I/R and hypoxia-reoxygenation (H/R) injury after GAS administration, respectively, to assess the impact of GAS on cardiomyocyte phenotypes, heart, and mitochondrial structure and function. The effect of GAS on cardiac function and mitochondrial structure in patients undergoing cardiac surgery has been observed in clinical practice. MethodsThe effects of GAS on cardiac structure and function, mitochondrial structure, and expression of related molecules in an animal model of MIRI were evaluated using immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), transmission electron microscopy, western blotting, and gene sequencing. Its effects on the morphological, molecular, and functional phenotypes of cardiomyocytes undergoing H/R were observed using immunohistochemical staining, real-time quantitative PCR, and western blotting. ResultsGAS significantly reduces myocardial infarct size and improves cardiac function in MIRI mice in animal models and increases cardiomyocyte viability and reduces cardiomyocyte damage in cellular models. In clinical practice, myocardial injury was alleviated with better cardiac function in patients undergoing cardiac surgery after the application of GAS; improvements in mitochondria and autophagy activation were also observed. GAS primarily exerts cardioprotective effects through activation of the PINK1/Parkin pathway, which promotes mitochondrial autophagy to clear damaged mitochondria. ConclusionGAS can promote mitophagy and preserve mitochondria through PINK1/Parkin, thus indicating its tremendous potential as an effective perioperative myocardial protective agent.

Peripheral Blood miRNA Expression in Patients with Essential Hypertension in the Han Chinese Population in Hefei, China.

Primary hypertension is a significant risk factor for cardiovascular diseases. However, the pathogenesis of primary hypertension involves multiple biological processes, including the nervous system, circulatory system, endocrine system, and more. Despite extensive research, there is no clear understanding of the regulatory mechanism underlying its pathogenesis. In recent years, miRNAs have gained attention as a regulatory factor capable of modulating the expression of related molecules through gene silencing. Therefore, exploring differentially expressed miRNAs in patients with essential hypertension (EH) may offer a novel approach for future diagnosis and treatment of EH. This study included a total of twenty Han Chinese population samples from Hefei, China. The samples consisted of 10 healthy individuals and 10 patients with EH. Statistical analysis was conducted to analyze the general information of the two-sample groups. High-throughput sequencing and base identification were performed to obtain the original sequencing sequences. These sequences were then annotated using various databases including Rfam, cDNA sequences, species repetitive sequences library, and miRBase database. The number of miRNA species contained in the samples was measured. Next, TPM values were calculated to determine the expression level of each miRNA. The bioinformatics of the differentiated miRNAs were analyzed using the OECloud tool, and RPM values were calculated. Furthermore, the reliability of the expression was analyzed by calculating the area under the Roc curve using the OECloud tools. Statistical analysis revealed no significant differences between the two samples in terms of age distribution, gender composition, smoking history, and alcohol consumption history (P > 0.05). However, there was a notable presence of family genetic history and high BMI in the EH population (P < 0.05). The sequencing results identified a total of 245 miRNAs, out of which 16 miRNAs exhibited differential expression. Among the highly expressed miRNAs were let-7d-5p, miR-101-3p, miR-122-5p, miR-122b-3p, miR-192-5p, and miR-6722-3p. On the other hand, the lowly expressed miRNAs included miR-103a-3p, miR-16-5p, miR-181a-2-3p, miR-200a-3p, miR-200b-3p, miR-200c-3p, miR-221-3p, miR-30d-5p, miR-342-5p, and miR-543. This study initially identified 16 miRNAs that are aberrantly expressed and function in various processes associated with the onset and progression of essential hypertension. These miRNAs have the potential to be targeted for future diagnosis and treatment of EH. However, further samples are required to provide additional support for this study.

Role of CTGF and PI3K/Akt signaling pathway in paraquat-induced mesenchymal changes in alveolar epithelial cells

Objective: To investigate the role of connective tissue growth factor (CTGF) and PI3K/Akt signaling pathways in paraquat (PQ) -induced alterations in alveolar epithelial cell mesenchymalization (EMT) . Methods: In February 2023, RLE-6TN cells were divided into 2 groups, which were set as uncontaminated group and contaminated group (200 μmol/L PQ), and cellular EMT alteration, CTGF and PI3K/Akt signaling pathway related molecules expression were detected by cell scratch assay, qRT-PCR and western-blot assay. Using shRNA interference technology to specifically inhibit the expression of CTGF, RLE-6TN cells were divided into four groups: control group, PQ group (200 μmol/L PQ), interference group (transfected with a plasmid with shRNA-CTGF+200 μmol/L PQ), and null-loaded group (transfected with a plasmid with scramble- CTGF+200 μmol/L PQ), qRT-PCR and western blot were used to examine the alteration of the cellular EMT and the expression of molecules related to the activity of PI3K/Akt pathway. The PI3K/Akt signaling pathway was blocked by the PI3K inhibitor LY294002, and the expression of EMT-related molecules in cells of the control group, PQ group (200 μmol/L PQ), and inhibitor group (200 μmol/L PQ+20 μmol/L LY294002) was examined by qRT-PCR and western blot.The t-test was used to compare the differences between the two groups, while the analysis of variance (ANOVA) was applied to compare the differences among multiple groups. For further pairwise comparisons, the Bonferroni method was adopted. Results: The results of cell scratch test showed that compared with the uncontaminated group, RLE-6TN cells in the contaminated group had faster migration rate, lower mRNA and protein expression levels of E-Cadherin, and higher mRNA and protein expression levels of α-SMA, CTGF, PI3K and Akt, with statistical significance (P<0.05). After specific inhibition of CTGF expression, the mRNA and protein expression of CTGF, PI3K, Akt, and α-SMA in the cells of the interference group were significantly lower than that of the PQ group and the null-loaded group (P<0.05/6), whereas that of E-Cadherin was higher than that of the PQ group and the null-loaded group (P<0.05/6). Specifically blocking the PI3K/Akt signaling pathway, the mRNA and protein expression of PI3K, Akt and α-SMA in the cells of the inhibitor group was decreased compared with that of the PQ group (P<0.05/3), while the expression of E-Cadherin was elevated compared with that of the PQ group (P<0.05/3) . Conclusion: CTGF may promote PQ-induced alveolar epithelial cell EMT through activation of the PI3K/Akt signaling pathway. Inhibition of CTGF expression or blockade of PI3K/Akt signaling pathway activity can alleviate the extent of PQ-induced alveolar epithelial cell EMT.

SIRT1 mediates the excitability of spinal CaMKIIα-positive neurons and participates in neuropathic pain by controlling Nav1.3.

Neuropathic pain is a common chronic pain disorder, which is largely attributed to spinal central sensitization. Calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα) activation in the spinal dorsal horn (SDH) is a major contributor to spinal sensitization. However, the exact way that CaMKIIα-positive (CaMKIIα+) neurons in the SDH induce neuropathic pain is still unclear. This study aimed to explore the role of spinal CaMKIIα+ neurons in neuropathic pain caused by chronic constriction injury (CCI) and investigate the potential epigenetic mechanisms involved in CaMKIIα+ neuron activation. CCI-induced neuropathic pain mice model, Sirt1loxP/loxP mice, and chemogenetic virus were used to investigate whether the activation of spinal CaMKIIα+ neurons is involved in neuropathic pain and its involved mechanism. Transcriptome sequence, western blotting, qRT-PCR, and immunofluorescence analysis were performed to assay the expression of related molecules and activation of neurons. Co-immunoprecipitation was used to observe the binding relationship of protein. Chromatin immunoprecipitation (ChIP)-PCR was applied to analyze the acetylation of histone H3 in the Scn3a promoter region. The expression of sodium channel Nav1.3 was increased and the expression of SIRT1 was decreased in the spinal CaMKIIα+ neurons of CCI mice. CaMKIIα neurons became overactive after CCI, and inhibiting their activation relieved CCI-induced pain. Overexpression of SIRT1 reversed the increase of Nav1.3 and alleviated pain, while knockdown of SIRT1 or overexpression of Nav1.3 promoted CaMKIIα+ neuron activation and induced pain. By knocking down spinal SIRT1, the acetylation of histone H3 in the Scn3a (encoding Nav1.3) promoter region was increased, leading to an increased expression of Nav1.3. The findings suggest that an aberrant reduction of spinal SIRT1 after nerve injury epigenetically increases Nav1.3, subsequently activating CaMKIIα+ neurons and causing neuropathic pain.

Expression and correlation analysis of related molecules in peripheral blood of patients with COVID-19

Expression and correlation analysis of related molecules in peripheral blood of patients with COVID-19

Citrulline facilitates the glycolysis, proliferation, and metastasis of lung cancer cells by regulating RAB3C.

Lung cancer (LC) is one of the major malignant diseases threatening human health. The study aimed to identify the effect of citrulline on the malignant phenotype of LC cells and to further disclose the potential molecular mechanism of citrulline in regulating the development of LC, providing a novel molecular biological basis for the clinical treatment of LC. The effects of citrulline on the viability, proliferation, migration, and invasion of LC cells (A549, H1299) were validated by CCK-8, colony formation, EdU, and transwell assays. The cell glycolysis was assessed via determining the glucose uptake, lactate production, ATP levels, extracellular acidification rate (ECAR), and oxygen consumption rate (OCR). RNA-seq and molecular docking were performed to screen for citrulline-binding target proteins. Western blotting experiments were conducted to examine the expression of related signaling pathway molecules. In addition, the impacts of citrulline on LC growth in vivo were investigated by constructing mouse models. Citrulline augmented the viability of LC cells in a concentration and time-dependent manner. The proliferation, migration, invasion, glycolysis, and EMT processes of LC cells were substantially enhanced after citrulline treatment. Bioinformatics analysis indicated that citrulline could bind to RAB3C protein. Western blotting results indicated that citrulline activated the IL-6/STAT3 pathway by binding to RAB3C. In addition, animal experiments disclosed that citrulline promoted tumor growth in mice. Citrulline accelerated the glycolysis and activated the IL6/STAT3 pathway through the RAB3C protein, consequently facilitating the development of LC.

Obeticholic acid attenuates the intestinal barrier disruption in a rat model of short bowel syndrome

BackgroundShort bowel syndrome (SBS) features nutrients malabsorption and impaired intestinal barrier. Patients with SBS are prone to sepsis, intestinal flora dysbiosis and intestinal failure associated liver disease. Protecting intestinal barrier and preventing complications are potential strategies for SBS treatment. This study aims to investigate the effects of farnesoid X receptor (FXR) agonist, obeticholic acid (OCA), have on intestinal barrier and ecological environment in SBS. Methods and resultsThrough testing the small intestine and serum samples of patients with SBS, impaired intestinal barrier was verified, as evidenced by reduced expressions of intestinal tight junction proteins (TJPs), increased levels of apoptosis and epithelial cell damage. The intestinal expressions of FXR and related downstream molecules were decreased in SBS patients. Then, global FXR activator OCA was used to further dissect the potential role of the FXR in a rat model of SBS. Low expressions of FXR-related molecules were observed on the small intestine of SBS rats, along with increased proinflammatory factors and damaged barrier function. Furthermore, SBS rats possessed significantly decreased body weight and elevated death rate. Supplementation with OCA mitigated the damaged intestinal barrier and increased proinflammatory factors in SBS rats, accompanied by activated FXR-related molecules. Using 16S rDNA sequencing, the regulatory role of OCA on gut microbiota in SBS rats was witnessed. LPS stimulation to Caco-2 cells induced apoptosis and overexpression of proinflammatory factors in vitro. OCA incubation of LPS-pretreated Caco-2 cells activated FXR-related molecules, increased the expressions of TJPs, ameliorated apoptosis and inhibited overexpression of proinflammatory factors. ConclusionsOCA supplementation could effectively ameliorate the intestinal barrier disruption and inhibit overexpression of proinflammatory factors in a rat model of SBS and LPS-pretreated Caco-2 cells. As a selective activator of FXR, OCA might realize its protective function through FXR activation.

Adenosine kinase inhibits β-cell proliferation by upregulating DNA methyltransferase 3A expression.

To investigate the effects of adenosine kinase (ADK), a key enzyme in determining intracellular adenosine levels, on β cells, and their underlying mechanism. Genetic animal models and transgenic immortalized cells were applied to study the effect of ADK on islet beta-cell proliferation and function. The beta-cell mass and response to glucose were measured in vivo using mice with beta-cell-specific ADK overexpression, and in vitro using ADK-overexpressed immortalized beta-cell. The expression of ADK in human islets at high abundance, especially in β cells, was decreased during the process of β-cell proliferation. Additionally, a transgenic mouse model (ADKtg/tg /Mip-Cre) was generated wherein the mouse Insulin1 gene promoter specifically overexpressed ADK in pancreatic β cells. The ADKtg/tg /Mip-Cre model exhibited impaired glucose tolerance, decreased fasting plasma insulin, loss of β-cell mass, and inhibited β-cell proliferation. Proteomic analysis revealed that ADK overexpression inhibited the expression of several proteins that promote cell proliferation and insulin secretion. Upregulating ADK in the β-cell line inhibited the expression of β-cell related regulatory molecules, including FoxO1, Appl1, Pxn, Pdx-1, Creb and Slc16a3. Subsequent in vitro experiments indicated that the inhibition of β-cell proliferation and the decreased expression of Pdx-1, Creb and Slc16a3 were rescued by DNA methyltransferase 3A (DNMT3A) knockdown in β cells. In this study, we found that the overexpression of ADK decreased the expression of several genes that regulate β cells, resulting in the inhibition of β-cell proliferation and dysfunction by upregulating the expression of DNMT3A.

MAPK4 facilitates angiogenesis by inhibiting the ERK pathway in non-small cell lung cancer.

Angiogenesis plays an important role in the occurrence and development of non-small cell lung cancer (NSCLC). The atypical mitogen-activated protein kinase 4 (MAPK4) has been shown to be involved in the pathogenesis of various diseases. However, the potential role of MAPK4 in the tumor angiogenesis of NSCLC remains unclear. Adult male C57BL/6 wild-type mice were randomly divided into the control group and p-siMAPK4 intervention group, respectively. The cell proliferation was analyzed withflow cytometry and immunofluorescence staining. The vascular density in tumor mass was analyzed by immunofluorescence staining. The expressions of MAPK4 and related signaling molecules were detected by western blot analysis and immunofluorescence staining, and so on. We found that the expression of MAPK4, which was dominantly expressed in local endothelial cells (ECs), was correlated with tumor angiogenesis of NSCLC. Furthermore, MAPK4 silencing inhibited the proliferation and migration abilities of human umbilical vein ECs (HUVECs). Global gene analysis showed that MAPK4 silencing altered the expression of multiple genes related to cell cycle and angiogenesis pathways, and that MAPK4 silencing increased transduction of the extracellular regulated protein kinases 1/2 (ERK1/2) pathwaybut not Akt and c-Jun n-terminal kinase pathways. Further analysis showed that MAPK4 silencing inhibited the proliferation and migration abilities of HUVECs cultured in tumor cell supernatant, which was accompanied with increased transduction of the ERK1/2 pathway. Clinical data analysis suggested that the higher expression of MAPK4 and CD34 were associated with poor prognosis ofpatients with NSCLC. Targeted silencing of MAPK4 in ECs using small interfering RNA driven by the CD34 promoter effectively inhibited tumor angiogenesis and growth of NSCLC in vivo. Our results reveal that MAPK4 plays an important role in the angiogenesis and development of NSCLC. MAPK4 may thus represent a new target for NSCLC.

Abstract 5573: Loss of glutathione S-transferase omega 2 promotes immunosuppressive tumor microenvironment by the induction of PD-L1 expression in lung adenocarcinoma

Abstract Glutathione S-transferase omega 2 (GSTO2) is known as one of important regulators for the glutathione redox balance. We recently reported that GSTO2 is exclusively expressed in airway basal cells, Clara cells, and type II alveolar cells, which have self-renewal capacity in the lungs; however, its expression was completely lost in lung squamous cell carcinoma (LSCC) (Cancer Science, 2022). In this study, we examined the expression of GSTO2 in lung adenocarcinoma (LAC), another type of lung cancer, and analyzed the relationship between GSTO2 expression and clinicopathological features. We enrolled 139 patients who were diagnosed with LAC and underwent surgery, and immunohistochemically evaluated the expression of GSTO2 using formalin-fixed, paraffin-embedded sections of surgical specimens. Fifty eight of 139 lung adenocarcinoma samples exhibited GSTO2 expression, while remaining 81 cases did not. The expression of GSTO2 in LAC samples was significantly associated with never or light (Brinkman Index &amp;lt; 400) smoking history (P=0.0027) and histological subtypes (lepidic 77%; acinar 44%; papillary 22%; micropapillary 16.7%; solid 9.6%; P&amp;lt;0.0001). There were no significant differences between the groups with respect to pN, pM, EGFR mutation, ALK fusion, and ROS-1 fusion. However, GSTO2 expression in LAC significantly associated with pT1 (P&amp;lt;0.0001), early stage (P&amp;lt;0.0001), and absence of PD-L1 expression (P=0.0027). Moreover, patients with GSTO2-negative LAC had a significantly lower disease-free survival rate than those with GSTO2-positve LAC (P=0.028). To examine whether GSTO2 expression affects PD-L1 expression, we prepared GSTO2-transfected and mock-transfected lung adenocarcinoma cell lines (A549 and PC-9). In both cell lines, mRNA level of PD-L1 was significantly reduced in GSTO2-transfectants. Because Jang RH et al reported that vimentin, reflecting to acquire mesenchymal traits in epithelial-mesenchymal transition (EMT), regulates the PD-L1 expression, we next examined whether GSTO2 overexpression affects the expression of EMT related molecules. There was no difference in a mRNA level of E-cadherin, while the mRNA expression of vimentin and SLUG, a transcriptional inducer of EMT, were significantly suppressed in GSTO2-transfected PC-9. In both cell lines, protein expression of vimentin was also significantly reduced in GSTO2-transfectants. The present study shed light on a novel function of GSTO2 as a PD-L1 regulator, which may contribute to prevention of tumor immune escape. Citation Format: Ryusuke Sumiya, Hiroto Hatano, Teruki Hagiwara, Satoshi Nagasaka, Kazuhiko Yamada, Norihiro Kokudo, Hiromu Suzuki, Yuki I. Kawamura. Loss of glutathione S-transferase omega 2 promotes immunosuppressive tumor microenvironment by the induction of PD-L1 expression in lung adenocarcinoma [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 5573.

Inhibiting Soluble Epoxide Hydrolase Suppresses NF-κB p65 Signaling and Reduces CXCL10 Expression as a Potential Therapeutic Target in Hashimoto's Thyroiditis.

Although Hashimoto's thyroiditis (HT) is one of most common autoimmune thyroid diseases, its treatment remains focused on symptom relief. The soluble epoxide hydrolase (sEH) shows potential functions as a drug target in alleviating some autoimmune diseases; however, we seldom know its role in HT. The protein expression of sEH and related downstream molecules were evaluated by immunohistochemistry, Western blotting, ELISA, or immunofluorescence staining. RNA sequencing of tissue samples was performed to analyze differential genes and dysregulated pathways in HT and controls. The thyroid follicular epithelial cells (TFECs) and rat HT model were used to verify the biological function of sEH and the inhibition role of adamantyl-ureido-dodecanoic acid (AUDA) in HT. The sEH was significantly upregulated in HT patients compared with healthy individuals. Transcriptome sequencing showed cytokine-related pathways and chemokine expression; especially chemokine CXCL10 and its receptor CXCR3 were aberrant in HT patients. In TFECs and a rat HT model, blocking sEH by AUDA inhibitor could effectively inhibit the autoantibody, proinflammatory nuclear kappa factor B (NF-κB) signaling, chemokine CXCL10/CXCR3 expression, and type-1 helper CD4+ T cells. Our findings suggest that sEH/NF-κB p65/CXCL10-CXCR3 might be promising therapeutic targets for HT.

Ablation of Shank1 Protects against 6-OHDA-induced Cytotoxicity via PRDX3-mediated Inhibition of ER Stress in SN4741 Cells.

Postsynaptic density (PSD) is an electron-dense structure that contains various scaffolding and signaling proteins. Shank1 is a master regulator of the synaptic scaffold located at glutamatergic synapses, and has been proposed to be involved in multiple neurological disorders. In this study, we investigated the role of shank1 in an in vitro Parkinson's disease (PD) model mimicked by 6-OHDA treatment in neuronal SN4741 cells. The expression of related molecules was detected by western blot and immunostaining. We found that 6-OHDA significantly increased the mRNA and protein levels of shank1 in SN4741 cells, but the subcellular distribution was not altered. Knockdown of shank1 via small interfering RNA (siRNA) protected against 6-OHDA treatment, as evidenced by reduced lactate dehydrogenase (LDH) release and decreased apoptosis. The results of RT-PCR and western blot showed that knockdown of shank1 markedly inhibited the activation of endoplasmic reticulum (ER) stress associated factors after 6-OHDA exposure. In addition, the downregulation of shank1 obviously increased the expression of PRDX3, which was accompanied by the preservation of mitochondrial function. Mechanically, downregulation of PRDX3 via siRNA partially prevented the shank1 knockdowninduced protection against 6-OHDA in SN4741 cells. In summary, the present study has provided the first evidence that the knockdown of shank1 protects against 6-OHDA-induced ER stress and mitochondrial dysfunction through activating the PRDX3 pathway.

Pth1r in Neural Crest Cells Regulates Nasal Cartilage Differentiation

Neural crest cells (NCC) arise from the dorsal margin of the neural plate border and comprise a unique cell population that migrates to and creates the craniofacial region. Although factors including Shh, Fgf8, and bone morphogenetic proteins have been shown to regulate these biological events, the role of parathyroid hormone 1 receptor (Pth1r) has been less studied. We generated an NCC-specific mouse model for Pth1r and researched gene expression, function, and interaction focusing on nasal cartilage framework and midfacial development. Wnt1-Cre;Pth1rfl/fl;Tomatofl/+ mice had perinatal lethality, but we observed short snout and jaws, tongue protrusion, reduced NCC-derived cranial length, increased mineralization in nasal septum and hyoid bones, and less bone mineralization at interfrontal suture in mutants at E18.5. Importantly, the mutant nasal septum and turbinate cartilage histologically revealed gradual, premature accelerated hypertrophic differentiation. We then studied the underlying molecular mechanisms by performing RNA seq analysis and unexpectedly found that expression of Ihh and related signaling molecules was enhanced in mutant nasomaxillary tissues. To see if Pth1r and Ihh signaling are associated, we generated a Wnt1-Cre; Ihh fl/fl; Pth1r fl/fl;Tomatofl/+ (DKO) mouse and compared the phenotypes to those of each single knockout mouse: Wnt1-Cre; Ihh fl/fl;Pth1rfl/+;Tomatofl/+ (Ihh-CKO) and Wnt1-Cre;Ihhfl/+; Pth1r fl/fl;Tomatofl/+ (Pth1r-CKO). Ihh-CKO mice displayed a milder effect. Of note, the excessive hypertrophic conversion of the nasal cartilage framework observed in Pth1r-CKO was somewhat rescued DKO embryos. Further, a half cAMP responsive element and the 4 similar sequences containing 2 mismatches were identified from the promoter to the first intron in Ihh gene. Gli1-Cre ERT2 ;Pth1rfl/fl;Tomatofl/+, a Pth1r-deficient model targeted in hedgehog responsive cells, demonstrated the enlarged hypertrophic layer and significantly more Tomato-positive chondrocytes accumulated in the nasal septum and ethmoidal endochondral ossification. Collectively, the data suggest a relevant Pth1r/Ihh interaction. Our findings obtained from novel mouse models for Pth1r signaling illuminate previously unknown aspects in craniofacial biology and development.

Clinical and pathological characteristics and expression of related molecules in patients with airway disseminated lung adenocarcinoma

Abstract Objective Lung adenocarcinoma exhibits diverse genetic and morphological backgrounds, in addition to considerable differences in clinical pathology and molecular biological characteristics. Among these, the phenomenon of spread through air space (STAS), a distinct mode of lung cancer infiltration, has rarely been reported. Therefore, this study aimed to explore the relationship between STAS tumor cells and the clinical and molecular characteristics of patients with lung adenocarcinoma, as well as their impact on prognosis. Methods This study included 147 patients who were diagnosed with lung adenocarcinoma at the Inner Mongolia Autonomous Region Cancer Institute between January 2014 and December 2017. Surgical resection specimens were retrospectively analyzed. Using univariate and multivariate Cox analyses, we assessed the association between STAS and the clinicopathological features and molecular characteristics of patients with lung adenocarcinoma. Furthermore, we investigated the effects on patient prognosis. In addition, we developed a column–line plot prediction model and performed internal validation. Results Patients with positive STAS had a significantly higher proportion of tumors with a diameter ≥2 cm, with infiltration around the pleura, blood vessels, and nerves, and a pathological stage &gt;IIB than in STAS-negative patients (P &lt; 0.05). Cox multivariate survival analysis revealed that clinical stage, STAS status, tumor size, and visceral pleural invasion were independent prognostic factors influencing the 5-year progression-free survival in patients with lung adenocarcinoma. The predictive values and P values from the Hosmer-Lemeshow test were 0.8 and 0.2, respectively, indicating no statistical difference. Receiver operating characteristic curve analysis demonstrated areas under the curve of 0.884 and 0.872 for the training and validation groups, respectively. The nomogram model exhibited the best fit with a value of 192.09. Conclusions Clinical stage, pleural invasion, vascular invasion, peripheral nerve invasion, tumor size, and necrosis are independent prognostic factors for patients with STAS-positive lung adenocarcinoma. The nomogram based on the clinical stage, pleural invasion, vascular invasion, peripheral nerve invasion, tumor size, and necrosis showed good accuracy, differentiation, and clinical practicality.

Euphorbia factorL2 suppresses the generation of liver metastatic ascites in breast cancer via inhibiting NLRP3 inflammasome activation.

Metastasis is the leading cause of death in patients with breast cancer, in part due to the lack of effective treatments. Euphorbia factorL2 (EFL2) is a diterpenoid extracted from Euphorbia lathyris L. seeds, which has attracted increasing attention in recent years due to its anticancer effect. However, the role and molecular mechanism of EFL2 in breast cancer liver metastasis remain unclear. In the present study, a breast cancer liver metastasis model was constructed and the effect of EFL2 on ascites generation in mice was examined. H&E staining detected inflammatory cells and tumor cells in the liver, small intestine and tumor tissues. Western blotting and reverse transcription‑quantitative PCR were used to detect the protein and mRNA expression of NLR family pyrin domain containing‑3 (NLRP3) and related molecules in tumor tissues. Immunohistochemistry was used to detect the levels of CD4 and CD8 Tcells in tumor tissue and immunofluorescence was used to further detect the expression level of NLRP3. Finally, the aforementioned experiments were further verified by overexpressing NLPR3. It was found that EFL2 inhibited generation of ascites in the model in a dose‑dependent manner. Furthermore, EFL2 inhibited tumor cell metastasis and enhanced immune cell infiltration. Meanwhile, EFL2 dose‑dependently downregulated the mRNA and protein expression of NLRP3 and related molecules in the model, and overexpression of NLRP3 abolished these beneficial effects of EFL2. Taken together, the present experimental data suggested that EFL2 has a significant inhibitory effect on ascites of breast cancer liver metastasis invivo, which may inhibit tumor cell metastasis by downregulating NLRP3 expression, providing an experimental basis for treating breast cancer metastasis.

Exosomes from adipose-derived stem cells restore fibroblast function and accelerate diabetic wound healing

BackgroundDiabetes is common yet challenging chronic disease, that affects a wide range of people around the world. Complex cellular environments around diabetic wounds tend to damage the function of effector cells, including vascular endothelial cells (VECs), fibroblasts and epithelial cells. This study aims to analyze the differences between diabetic wounds and normal skin as well as whether adipose-derived stem cell (ADSC) exosome could promote healing of diabetic wound. MethodsHuman diabetic wounds and normal skin were collected and stained with HE, Masson, CD31 and 8-hydroxy-2 deoxyguanosine immunohistochemical staining. RNA-seq data were collected for further bioinformatics analysis. ADSC exosomes were isolated and identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting. The effect of ADSC exosomes on diabetic wound healing was assessed on full thickness wounds in mice. To further verify the regulative impact of ADSCs exosomes in high glucose treated fibroblasts, we isolated fibroblasts from normal skin tissue and measured the cell viability, apoptosis rate, proliferation and migration of fibroblasts. In addition, collagen formation and fibrosis-related molecules were also detected. To further disclose the mechanism of ADSC exosomes on the function of high glucose treated fibroblasts, we detected the expression of apoptosis related molecules including BCL2, Bax, and cleaved caspase-3. ResultsHistological observation indicated that perilesional skin tissues from diabetic patients showed structural disorder, less collagen disposition and increased injury compared with normal skin. Bioinformatics analysis showed that the levels of inflammatory and collagen synthesis related molecules, as well as oxidative stress and apoptosis related molecules, were significantly changed. Furthermore, we found that ADSC exosomes could not only speed up diabetic wound healing, but could also improve healing quality. ADSC exosomes restored high glucose induced damage to cell viability, migration and proliferation activity, as well as fibrosis-related molecules such as SMA, collagen 1 and collagen 3. In addition, we verified that ADSC exosomes downregulated high glucose induced increased apoptosis rate in fibroblast and the protein expression of Bax as well as cleaved caspases 3. ConclusionsThis study indicated that ADSC exosomes alleviated high glucose induced damage to fibroblasts and accelerate diabetic wound healing by inhibiting Bax/caspase 3.

Deficiency of SR-B1 reduced the tumor load of colitis-induced or APCmin /+ -induced colorectal cancer.

Colorectal cancer (CRC) is one of the most common tumors in the world. Cholesterol plays an important role in the pathogenesis of tumors. One of the cholesterol transporters, scavenger receptor class B type 1 (SR-B1), a multi-ligand membrane receptor protein, is expressed in the intestines which also highly expressed in various tumors. But the potential mechanism of SR-B1 in CRC development has not been reported. This study aimed to clarify the importance of SR-B1 in the development and prognosis of CRC as much as possible to provide a possible strategy in CRC treatment. In this study, we used SR-B1 gene knockdown mice to study the effect of SR-B1 on colitis-induced or APCmin/+ -induced CRC. The expression of related molecules were detected through the immunohistochemistry and hematoxylin-eosin staining, western blot analysis, and Flow cytometry. The gene expression and microbiota in microenvironment of CRC mice were analyzed through eukaryotic mRNA sequencing and 16S rRNA high-throughput sequencing. The results showed that SR-B1 knockdown reduced the tumor load of colitis-induced or APCmin/+ -induced CRC. SR-B1 knockdown improved the immune microenvironment by affecting the level of tumor-associated macrophage (TAM), mononuclear myeloid-derived suppressor cells (M-MDSCs), granulocytic myeloid-derived suppressor cells (G-MDSCs), programmed cell death-ligand 1 (PD-L1), and human leukocyte antigen class I-B (HLA-B), and also reduced the level of low-density lipoprotein receptor (LDL-R), and increased the level of ATP binding cassette transporter A1 (ABCA1) to regulate the cholesterol metabolism, and regulated the expression of related genes and intestinal microbiota. SR-B1 knockdown can also trigger the anti-CRC effect of anti-PD 1 in colitis-induced CRC. SR-B1 deficiency significantly improved the immunity in tumor microenvironment of colitis-induced or APCmin/+ -induced CRC. In addition, the microbiota changes caused by SR-B1 deficiency favor improving the immune response to chemotherapeutic drugs and anti-PD1 therapy. The mechanism of action of SR-B1 deficiency on the development of CRC still needs further in-depth research. This study provides a new treatment strategy for treating CRC by affecting the expression of SR-B1 in intestine.

Expression Pattern of Immune-Related Genes in the Liver of Rohu, &lt;i&gt;Labeo rohita&lt;/i&gt; (Hamilton) Exposed to Carbon Tetrachloride Toxicity

Immune responses in the liver play a crucial role in the detoxification process and renderingprotection against different diseases. The present study was targeted to analyze the expressionkinetics of few immune genes (pro-inflammatory, immune receptors, anti-microbial and innateimmunity-related) using qPCR in liver tissue of carbon tetrachloride (CCl4) treated rohu (Labeorohita). Rohu juveniles were injected with single intraperitoneal toxic dose of 30% CCl4 to induce liver damage. Histopathological findings showed the presence of focal necrosis and vaccuolation of hepatocytes. Increase in the level of pro-inflammatory cytokine (interleukin-6, IL-8 and IL-15) transcripts was observed immediately after CCl4 post-exposure (from 6 h to 120 h), thus indicating the role of enhanced level of cytokines inducing liver damage. The degree of liver damage was also reflected by reduction in expression of immune related molecules (viz., TLR 22, transferrin and hepcidin), which are mostly synthesized in liver tissue. No effect was noticed in synthesis of mitochondrial antiviral-signaling protein (MAVS) transcripts with the current level of CCl4 exposure. These results suggest that there is a critical balance between immune molecules that may play essential roles in the orchestration of immune defense in fish during liver dysfunction.