Expression Of Related Factors Research Articles

Saikosaponin-d regulates angiogenesis in idiopathic pulmonary fibrosis through angiopoietin/Tie-2 pathway

ObjectiveIdiopathic pulmonary fibrosis (IPF) is considered as a chronic interstitial lung disease with underlying mechanism of IPF remaining unclear, while there are no definitive treatment options. In recent years, scientists have gradually paid attention to the influence of angiogenesis on IPF. Because IPF is a progressive with microvascular remodeling disorder, scientists have postulated that angiogenesis may also be one of the initiating and contributing factors of the disease. Bupleurum is a common natural Chinese herbal medicine with antibacterial, anti-inflammatory, anti-tumor and other pharmacological effects. As the most important active monomer of Bupleurum, Saikosaponin-d (SSd) is a new discovery with anti-pulmonary fibrosis (PF) activity. This study attempts to investigate the role of SSd in the interference of PF through regulation of angiogenesis in IPF through Angiopoietin (Angpt) /Tie receptor 2 (Tie2) pathway. MethodsRandomly, we allocated C57BL/6 mice into four groups (n = 20 in each group). Afterwards, establishment of IPF model was accomplished via intratracheal administration of bleomycin (BLM, 5 mg/kg), while corresponding drug intervention was given accordingly. On 3rd, 7th, 14th and 28th days after modeling, we performed histopathological examination through staining. Meanwhile, immunohistochemistry (IHC) of PF and the expression of related factors were observed, while Ang/Tie2 pathway was assessed by ELISA with the effect of SSd on angiogenesis related proteins in IPF being explored with IHC and Western Blot technique. ResultsOur results showed that SSd could reduce inflammation and PF levels in lung tissue of experimental mice, while levels of angiogenesis-related factors, namely Tie-2, Ang-1 and ANGPT2 (Ang-2), fibrosis- associated factors like Alpha-smooth muscle actin (α-SMA), collagen-I and hydroxyproline in SSd and dexamethasone (DXM) mice were significantly reduced at each time point compared to BLM (p < 0.01). Additionally, we discovered substantial decreased expressions of Ang-1, Ang-2, Tie-2, α-SMA and collagen-I at protein level in SSd and DXM mice at each time point compared to BLM (p < 0.05). Besides, insignificant differences were observed between SSd and DXM groups (p > 0.05). ConclusionThis study has demonstrated that SSd could down-regulate the expression of ANG-1, Ang-2 and Tie2 in the Ang/Tie2 pathway, and may reduce lung inflammation and PF in BLM-induced mice via inhibition of angiogenesis.

The role of phosphoprotein associated with glycosphingolipid-enriched microdomains 1 (PAG1) in regulating the progression of oral squamous cell carcinoma

ObjectiveThe aim of this study was to explore the role of the tumor suppressor phosphoprotein associated with glycosphingolipid-enriched microdomains 1 (PAG1) on oral squamous cell carcinoma (OSCC) and its molecular mechanism. DesignImmunohistochemistry detected the expression of PAG1 in normal and tumor tissues. The PAG1 overexpressed OSCC cell lines were constructed by lentivirus transfection. Cell Counting Kit-8 assay (CCK-8), clone formation and flow cytometry evaluated the impact of PAG1 on the proliferation and apoptosis of OSCC cells. RNA sequencing (RNA-seq) detected the changes in intracellular genes, and transmission electron microscope (TEM) was used to compare the number of autophagosomes in OSCC cells between Negative and PAG1 group. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and Western blot were used to determine the expression of signaling pathway-related mRNA and proteins respectively. ResultsIn contrast to the normal tissues, PAG1 expression was significantly downregulated in tumor tissues. Treatment with lentivirus transfection, the expression of PAG1 in the OSCC cell lines was increase. Notably, transfected with PAG1-overexpressing lentivirus cells inhibited the proliferation of OSCC cells and promoted OSCC cells apoptosis. RNA-seq revealed that PAG1 mainly modulated the mitophagy and autophagy pathway, and many autophagosomes were observed in the PAG1 group using TEM. Mechanistically, we found that PAG1 upregulated the expression of autophagy related factors through inhibiting PI3K/Akt/mTOR signal pathway activation. ConclusionOverexpression of PAG1 inhibited OSCC progression by activating autophagy, its mechanism might be related to inhibition of PI3K/Akt/mTOR signal pathway phosphorylation.

RUNX1 as a Potential Target for Combined Radioimmunotherapy of Lung Adenocarcinoma

Radioimmunotherapy for non-small cell lung cancer has good clinical application prospects. The role and mechanism of RUNX1 in DNA damage repair were explored for its potential role in lung adenocarcinoma radioimmunotherapy. To study the effect of RUNX1 expression level on the expression of DNA damage repair system related factors and radiation sensitivity of lung adenocarcinoma cells. As an important nuclear transcription factor, RUNX1 was explored whether directly regulating the expression of Nrf2, Rad51, BRCA1, and verifying their respective DNA binding sites in the promoter region through relevant databases. To observe the effect of RUNX1 knockout and overexpression on the expression level of PD-L1 in tumor cells at the cell level; The effect of RUNX1 expression level on the sorting and presentation of PD-L1 cells was investigated by the method of nucleocytoplasmic separation. According to literature reports, CMTM6 and ALIX play a key role in the process of PD-L1 cell sorting and presentation, and explore whether RUNX1 plays a role through this factor. The effect of phosphorylation level of different splicing bodies of RUNX1 (RUNX1a/b/c) on the expression level and DNA damage repair system related factors on tumor radiosensitivity were also explored. According to TCGA database, RUNX1 is highly expressed and phosphorylated in lung adenocarcinoma. Through gene comparison with the database, it was found that RUNX1 binding sites existed in the promoter region of several factors related to this study, including ALIX, Nrf2, BRCA1, RAD51, ATM, H2AX, etc. After being activated by MAPKp38 phosphorylation, RUNX1a can positively regulate Nrf2 signal pathway. The expression of RUNX1 and p-RUNX1 is time-dependent on ionizing radiation. At the same time, it was found that the expression of RUNX1 and p-RUNX1 was dose-dependent on ionizing radiation, and the expression trend of Nrf2 signal pathway related factors was consistent with RUNX1. RUNX1 regulates the expression of PD-L1, BRCA1, ALIX and Nrf2. Bioinformatics analysis and flow cytometry data show that RUNX1 has inhibitory effect on tumor microenvironment of lung adenocarcinoma. RUNX1 regulates DNA damage repair system and has inhibitory effect on tumor immunity. Inhibiting the expression of RUNX1 in lung adenocarcinoma cells can enhance the effect of radioimmunotherapy.

Study on the effect and mechanism of verapamil on the growth of human airway granulosa fibroblasts

Objective: This study aimed to investigate the inhibitory effect of the calcium channel blocker verapamil on human airway sarcomeric fibroblasts through network pharmacology molecular docking combined with experimental validation, so as to provide a theoretical and experimental basis for clinical use of calcium channel blockers against benign airway stenosis. Methods: The relevant targets of verapamil, airway injury, sarcoidosis and fibroblast were collected and intersected by a public database, respectively. The protein-protein interaction network was constructed by combining STRING; the DAVID database was used for gene function GO and KEGG pathway enrichment analysis. Molecular docking validation of verapamil and key targets was performed using AutoDock. Meanwhile, human lung bronchial tissue and human airway granulation tissue were treated with different antibiotic regimens to prevent contamination, in which human normal airway fibroblasts were treated with 92.81 μg/ml verapamil, while airway granulosa fibroblasts were treated with 69.57 μg/ml verapamil. The flow cytometry test was performed to detect the apoptotic effect of verapamil on human airway granulosa fibroblasts. The relative expression of mRNA of relevant factors between human normal airway fibroblasts and human airway granulosa fibroblasts was detected using polymerase chain reaction (PCR). Meanwhile, the difference in mRNA expression between human airway fibroblasts in normal and disease conditions was measured by PCR, and the change in mRNA expression of related factors after treatment with verapamil was determined to verify the findings of network pharmacology analysis. Results: This study predicted that verapamil affects 162 targets in human airway granulosa fibroblasts, among which, the most concerned and core target proteins are TGF-β1, VEGFA and IL-6; mainly involved in biological processes such as apoptosis, angiogenesis, energy metabolism and inflammation, and AGE-RAGE, JAK-STAT, PI3K-AKT, HIF as well as IL-17 signaling pathways. Molecular docking results showed that verapamil can bind to key targets. The apoptosis rate of human airway granulomatous fibroblasts treated with verapamil was higher than that of the control group, and the difference was statistically significant (P &lt; 0.05). PCR showed that the relative expression of TGF-β1, VEGFA, IL-6 and other mRNAs in human airway granulomatous fibroblasts was significantly higher than that in human normal airway fibroblasts (P &lt; 0.05), whereas the relative expression of TGF-β1 mRNA was down-regulated (P &lt; 0.05) and the mRNA expression of VEGFA, IL-6 did not change significantly (P &gt; 0.05) in human airway granulosa fibroblasts after treatment with verapamil. Conclusions: Increasing the antibiotic concentration and combining multiple antibiotics to soak the tissue can effectively kill the tissue pathogens. Verapamil can inhibit the proliferation of airway granulomatous fibroblasts through multiple targets and pathways, and it may be a method to prevent and treat benign airway stenosis.

Reactive Oxygen Species Scavenging Hydrogel Regulates Stem Cell Behavior and Promotes Bone Healing in Osteoporosis.

Implantation of bone marrow mesenchymal stem cells (BMSCs) is a potential alternative for promoting bone defects healing or osseointegration in osteoporosis. However, the reactive oxygen species (ROS) accumulated and excessive inflammation in the osteoporotic microenvironment could weaken the self-replication and multi-directional differentiation of transplanted BMSCs. In this study, to improve the hostile microenvironment in osteoporosis, Poloxamer 407 and hyaluronic acid (HA) was crosslinked to synthetize a thermos-responsive and injectable hydrogel to load MnO2 nanoparticles as a protective carrier (MnO2@Pol/HA hydrogel) for delivering BMSCs. The resulting MnO2@Pol/HA hydrogel processed excellent biocompatibility and durable retention time, and can eliminate accumulated ROS effectively, thereby protecting BMSCs from ROS-mediated inhibition of cell viability, including survival, proliferation, and osteogenic differentiation. In osteoporotic bone defects, implanting of this BMSCs incorporated MnO2@Pol/HA hydrogel significantly eliminated ROS level in bone marrow and bone tissue, induced macrophages polarization from M1 to M2 phenotype, decreased the expression of pro-inflammatory cytokines (e.g., TNF-α, IL-1β, and IL-6) and osteogenic related factors (e.g., TGF-β and PDGF). This hydrogel-based BMSCs protected delivery strategy indicated better bone repair effect than BMSCs delivering or MnO2@Pol/HA hydrogel implantation singly, which providing a potential alternative strategy for enhancing osteoporotic bone defects healing.

Modulatory effects of point-mutated IL-32θ (A94V) on tumor progression in triple-negative breast cancer cells.

Breast cancer is a frequently diagnosed cancer and the leading cause of death among women worldwide. Tumor-associated macrophages stimulate cytokines and chemokines, which induce angiogenesis, metastasis, proliferation, and tumor-infiltrating immune cells. Although interleukin-32 (IL-32) has been implicated in the development and modulation of several cancers, its function in breast cancer remains elusive. Mutation of interleukin-32θ (IL-32θ) in the tissues of patients with breast cancer was detected by Sanger sequencing. RT-qPCR was used to detect the mRNA levels of inflammatory cytokines, chemokines, and mediators. The secreted proteins were detected using respective enzyme-linked immunosorbent assays. Evaluation of the inhibitory effect of mutant IL-32θ on proliferation, migration, epithelial-mesenchymal transition (EMT), and cell cycle arrest in breast cancer cells was conducted using MTS assays, migration assays, and Western blotting. A point mutation (281C>T, Ala94Val) was detected in IL-32θ in both breast tumors and adjacent normal tissues, which suppressed the expression of pro-inflammatory factors, EMT factors, and cell cycle related factors. Mutated IL-32θ inhibited the expression of inflammatory factors by regulating the NF-κB pathway. Furthermore, mutated IL-32θ suppressed EMT markers and cell cycle related factors through the FAK/PI3K/AKT pathway. It was inferred that mutated IL-32θ modulates breast cancer progression. Mutated IL-32θ (A94V) inhibited inflammation, EMT, and proliferation in breast cancer by regulating the NF-κB (p65/p50) and FAK-PI3K-GSK3 pathways.

Matrine inhibits inflammatory response induced by TNF-α in human umbilical vein endothelial cells through miR-25-3p-mediated Klf4 pathway

This study aimed to analyze the effect of matrine on tumor necrosis factor-α(TNF-α)-induced inflammatory response in human umbilical vein endothelial cells(HUVECs) and explore whether the underlying mechanism was related to the miR-25-3p-mediated Krüppel-like factor 4(Klf4) pathway. The HUVEC cell inflammation model was induced by TNF-α stimulation. After 24 or 48 hours of incubation with different concentrations of matrine(0.625, 1.25, and 2.5 mmol·L~(-1)), CCK-8 assay was used to detect cell proliferation. After treatment with 2.5 mmol·L~(-1) matrine for 48 h, the expression of TNF-α, interleukin-6(IL-6), interleukin-1β(IL-1β), and Klf4 mRNA and miR-25-3p was detected by real-time fluorescence-based quantitative PCR, and the protein expression of TNF-α, IL-6, IL-1β, and Klf4 was detected by Western blot. The anti-miR-25-3p was transfected into HUVECs, and the effect of anti-miR-25-3p on TNF-α-induced cell proliferation and inflammatory factors was detected by the above method. The cells were further transfected with miR-25-3p and incubated with matrine to detect the changes in proliferation and expression of related inflammatory factors, miR-25-3p, and Klf4. The targeting relationship between miR-25-3p and Klf4 was verified by bioinformatics analysis and dual luciferase reporter gene assay. The results displayed that matrine could inhibit TNF-α-induced HUVEC proliferation, decrease the mRNA and protein expression of TNF-α, IL-6, and IL-1β, increase the mRNA and protein expression of Klf4, and reduce the expression of miR-25-3p. Bioinformatics analysis showed that there were specific complementary binding sites between miR-25-3p and Klf4 sequences. Dual luciferase reporter gene assay confirmed that miR-25-3p negatively regulated Klf4 expression in HUVECs by targeting. The inhibition of miR-25-3p expression can reduce TNF-α-induced cell proliferation and mRNA and protein expression of TNF-α, IL-6, and IL-1β. MiR-25-3p overexpression could reverse the effect of matrine on TNF-α-induced cell proliferation and the mRNA and protein expression of TNF-α, IL-6, IL-1β, and Klf4. This study shows that matrine inhibits the inflammatory response induced by TNF-α in HUVECs through miR-25-3p-mediated Klf4 pathway.

Melatonin alleviates 3,3',4,4',5-pentachlorobenzene induced colon injury by relieving oxidative stress.

3,3',4,4',5-pentachlorobiphenyl (PCB126) is widely distributed, non-degradable and bioaccumulative, which can affect the function of tissues and organs of the living organisms. Melatonin (MT) is a sort of indole neurohormone that is mainly secreted by the pineal gland. Numerous studies have shown that MT can alleviate intestinal injury through various mechanisms such as antioxidant, anti-inflammatory, and anti-apoptosis. For the above reasons, the aim of this study is to explore the mechanism of intestinal injury in mice after exposure to PCB126 as well as the antagonistic effect of MT. Mice were respectively fed PCB126 (0.326 mg/kg) and/or MT (10 mg/kg) in vivo. In vitro, colonic epithelial cells (MCEC) were treated with PCB126 (150 μM) and/or MT (2 mM). We found that the microscopic structure of colon tissue was impaired after exposure to PCB126. The levels of oxidative stress, the protein and mRNA levels of expression of inflammatory related factors were significantly increased and the expression levels of intestinal tight junction protein were decreased. Notably, MT can promote Nrf2/HO-1 expression level and reduce the colonic injury caused by PCB126. Further in vitro treatment with reactive oxygen species inhibitors (NAC) showed that it significantly alleviated PCB126-induced in MCEC cell damage. In summary, the above results suggested that MT alleviates PCB126-induced colon inflammation by inhibiting the overproduction of reactive oxygen species (ROS) and up-regulating the expression level of intestinal tight junction protein. Our results contribute to the further comprehension of the intestinal toxicity effects of PCB126 and the significant role of MT in preserving the mechanisms of intestinal injury.

LINC01116 Regulates the Proliferation and Apoptosis of Nucleus Pulposus Cells through miR-9-5p-mediated ZIC5 and the Wnt Pathway and Affects the Progression of Intervertebral Disc Degeneration.

Intervertebral disc degeneration (IDD) represents one of the leading causes of low back pain. Research suggests the participation of LINC01116 in IDD progression. Herein, the current study explored the underlying mechanism of LINC01116 in IDD. The differential expression patterns of LINC01116 in IDD and normal tissues were analyzed using the GEO database. Human nucleus pulposus (NP) cells were provided and treated with IL-1β to establish IDD models in vitro. LINC01116 expression was detected and intervened. Indices such as cell proliferation, apoptosis, and extracellular matrix (ECM)-related factor expression were determined using CCK-8 assay, flow cytometry, and Western blotting. LINC01116 sublocation was identified by means of nuclear/cytosol fractionation assay. The binding relationships between LINC01116 and miR-9-5p and miR-9-5p and ZIC5 were verified by bioinformatics analysis, dual-luciferase assays, RNA immunoprecipitation (RIP) assay, and RNA-pull-down. Western blotting was conducted to measure the levels of the Wnt pathway key factors. LINC01116 was highly expressed in the degenerative NP cells. Silencing of LINC01116 critically promoted degenerative NP cell proliferation and inhibited apoptosis and ECM loss. LINC01116 was located in the cytoplasm. In degenerative NP cell models, LINC01116 could competitively bind to miR-9-5p to elevate ZIC5 expression. LINC01116 induced NP cell apoptosis and impeded NP cell proliferation and ECM synthesis by inhibiting miR-9-5p and miR-9-5p targeted ZIC5. ZIC5 could effectively increase the levels of the Wnt pathway-related factors. Silencing LINC01116 blocked its adsorption of miR-9-5p as a sponge to promote the miR-9- 5p expression and inhibit ZIC5/Wnt activation, thus impacting NP cell biological functions.

A comparative metabolomics study between grain-sized moxibustion and suspended moxibustion on rats with gastric ulcers

Grain-sized moxibustion (GS-Moxi) and suspended moxibustion (S-Moxi) represent the two typical local heat therapies in Traditional Chinese Medicine (TCM) and have been extensively used in treating gastric ulcers (GU) in China. However, the difference in biological response between the two moxibustion therapies in treating GU remains unclear. Here we investigated the therapeutic effect and potential mechanistic difference underlying the two moxibustion methods. Ethanol-induced GU model was established and was treated with GS-Moxi or S-Moxi at ST36 and ST21 for 5 days separately. And then, gastric histopathological examination, immunohistochemical staining for repair factors (EGFR, VEGF, Ki67), and 1H NMR-based metabolomics analysis of plasma and stomach of rats were conducted. We found GS-Moxi and S-Moxi effectively alleviated gastric damage and significantly increased the expression of related repair factors. However, S-Moxi corrected aberrant energy metabolism and lipids metabolism in GU rats but had little effect on neurotransmitter-related metabolism, while GS-Moxi regulated energy metabolism and neurotransmitter-related metabolism in GU rats but had no effect on lipids metabolism. We further proposed that the main target of S-Moxi may be liver and vasculature, whereas GS-Moxi specially targeted the stomach via regulating nervous system. This study strongly verified the outstanding gastroprotective effects of moxibustion and enriched our understanding of the varied biological responses triggered by different moxibustion methods.

ADAMTS5 promotes neovascularization via autophagic degradation of PEDF in proliferative diabetic retinopathy

Proliferative diabetic retinopathy (PDR) adversely affects visual function. Extracellular matrix proteins (ECM) contribute significantly to the development of PDR. A Disintegrin and Metalloproteinase with Thrombospondin motifs 5 (ADAMTS5) is a member of ECM proteins. ADAMTS5 participates in angiogenesis and inflammation in diverse diseases. However, the role of ADAMTS5 in PDR remains elusive. Multiplex beam array technology was used to analyze vitreous humor of PDR patients and normal people. ELISA and Western blot were used to detect the expression of ADAMTS5, PEDF and autophagy related factors. Immunofluorescence assay was used to mark the expression and localization of ADAMTS5 and PEDF. The neovascularization was detected by tube formation test. Our results revealed that ADAMTS5 expression was increased in the vitreous humor of PDR patients and oxygen-induced retinopathy (OIR) mice retinas. Inhibiting ADAMTS5 alleviated pathological angiogenesis and upregulated PEDF expression in the OIR mice. In addition, ADAMTS5 inhibited PEDF secretion in ARPE-19 cells in vitro studies, thereby inhibiting the migration of HMEC-1. Mechanically, ADAMTS5 promoted the autophagic degradation of PEDF. Collectively, inhibition of ADAMTS5 during OIR suppresses pathological angiogenesis. Our study provides a new approach for resolving pathological angiogenesis in PDR.

Dyslipidemia and Thyroid Dysfunction Associated with Psoriasis.

Psoriasis is a T-cell mediated inflammatory skin disorder which causes considerable psychosocial disability resulting in major impact on patient's quality of life. The dominant response of Th1 cells and the high expression of related inflammatory factors appears in both psoriasis and thyroid dysfunction. Psoriasis can lead to increased risk of incidence of cardiovascular events for which dyslipidemia is an important risk factor. This study aims to evaluate thyroid function and lipid profile and its association in patients with psoriasis. This was a prospective case-control study conducted at the Department of Biochemistry, Nobel Medical College Teaching Hospital from November 2021 to October 2022. Seventy six clinically diagnosed psoriatic patients were taken as case and eighty non-psoriatic patients coming for other skin lesions were included in the study as control. Thyroid function test (serum free T3, T4, TSH) and lipid profile (total cholesterol, low density lipoprotein, high density lipoprotein and triglyceride) were done. Hypothyroidism was most common thyroid abnormalities in our study population which was 13.15% (n=10). The lipid profile parameters were significantly higher in the psoriatic patients than in the controls (p<0.001). The odds of psoriatic patient having thyroid dysfunction was 2.8 times higher compared to other non-psoriatic patients and the odds of psoriatic patient having dyslipidemia was 8.7 times higher compared to other non-psoriatic patients. It is thus useful to assess thyroid function test and lipid profile in patients with psoriasis considering their role in etiopathogenesis and co-morbidity of psoriasis.

Anti-inflammatory material basis and mechanism of Artemisia stolonifera based on UPLC-Q-TOF-MS combined with network pharmacology and molecular docking

This study aimed to explore the anti-inflammatory material basis and molecular mechanism of Artemisia stolonifera based on the analysis of the chemical components in different extracted fractions of A. stolonifera and their antioxidant and anti-inflammatory effects in combination with network pharmacology and molecular docking. Thirty-two chemical components were identified from A. stolonifera by ultra-performance liquid chromatography coupled to tandem quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS). Among them, there were 7, 21 and 22 compounds in water, n-butanol and ethyl acetate fractions, respectively. The antio-xidant capacity of different extracted fractions was evaluated by measuring their scavenging ability against 1,1-diphenyl-2-picrylhydrazyl radical 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl(DPPH) and 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonic acid)(ABTS) free radicals and total antioxidant capacity [ferric reducing antioxidant power(FRAP) assay]. The inflammatory model of RAW264.7 cells was induced by lipopolysaccharide(LPS), and the levels of nitrite oxide(NO), tumor necrosis factor-α(TNF-α), interleukin-6(IL-6) in the supernatant and the mRNA expression of related inflammatory factors in cells were used to evaluate the anti-inflammatory effects. The results revealed that ethyl acetate fraction of A. stolonifera was the optimal antioxidant and anti-inflammatory fraction. By network pharmacology, it was found that flavonoids such as rhamnazin, eupatilin, jaceosidin, luteolin and nepetin could act on key targets such as TNF, serine/threonine protein kinase 1(AKT1), tumor protein p53(TP53), caspase-3(CASP3) and epidermal growth factor receptor(EGFR), and regulate the phosphatidylinositol-3-kinase-protein kinase B(PI3K-AKT) and mitogen-activated protein kinase(MAPK) signaling pathways to exert the anti-inflammatory effects. Molecular docking further indicated excellent binding properties between the above core components and core targets. This study preliminarily clarified the anti-inflammatory material basis and mechanism of ethyl acetate fraction of A. stolonifera, providing a basis for the follow-up clinical application of A. stolonifera and drug development.

Prostanoid FP and EP2 Receptor Agonists Induce Epithelial and Subepithelial Fibrogenetic Changes in Human Conjunctival Fibroblasts in Different Manners.

Purpose: To examine the effects of prostanoid FP and EP2 receptor agonists, PGF2α and Omidenepag (OMD), respectively, on the transforming growth factor beta (TGF-β2) induced conjunctival fibrogenesis. Methods: Two-dimension (2D) and three-dimension (3D) cultures of these fibroblasts were subjected to following analyses: (1) planar proliferation evaluated by transendothelial electron resistance (TEER) measurements, (2) real-time metabolic analyses, (3) subepithelial proliferation evaluated by 3D spheroid' size and stiffness measurements, and (4) the mRNA expression of extracellular matrix (ECM) molecules and their modulators. Results: TGF-β2 induced increase in the planar proliferation was significantly decreased or enhanced by PGF2α or OMD, respectively. The proportion of oxygen consumption required to drive ATP synthesis compared with that driving proton leakage was increased by PGF2α and OMD independently with TGF-β2. In contrast, maximal mitochondrial respiration was decreased by PGF2α and OMD, and the OMD-induced effect was further enhanced by the presence of TGF-β2. In addition, the TGF-β2 dependent increase in the glycolytic capacity was cancelled by PGF2α and/or OMD. Alternatively, subepithelial proliferation, as evidenced by the stiffness of the 3D spheroids, was substantially increased by both PGF2α and OMD, and these were differently modulated by TGF-β2. The expression of several related factors as above fluctuated among the conditions for both 2D and 3D and TGF-β2 untreated or treated cultures. Conclusion: The present findings indicate that the prostanoid FP or the EP2 receptor agonist may solely and differently induce the planar and subepithelial proliferation of HconF cells and these were also modulated by TGF-β2.

Study of hydrosalpinx on endometrial growth and expression of HOXA10mRNA and related factors

ObjectiveThe objective of the study is to extract the patient's endometrium at the time of proliferative stage using hydrosalpinx in order to culture the cells and decidualization induction in vitro. Further, the study is also intended at identifying the expression of HOXA10mRNA and related factors and understand the hydrosalpinx's impact upon the working mechanism of endometrial cells. MethodsOnce the extraction of the primary cells is over, the cells are cultured and other activities are performed such as the cell identification, CCK8 assay, cell decidua induction and HE staining. The researchers assessed the expression levels of HOXA10, IGFBP1 and avβ3 in either proliferation or secretion of the endometrium. This was accomplished using Western blot assay and real-time fluorescence quantitative PCR. ResultsThe results confirmed that at the time of endometrial proliferation, there was a decline in the expression of HOXA10 as a result of HS influence. This affected its expression in the secretory stage i.e., corresponding function. Further, a significant decline was observed in the levels of HOXA10mRNA of endometrial cells that were subjected to continuous HS, post decidualization. It was found that during decidualization, if the HS is removed, it is possible to restore the expression of HOXA10mRNA to a certain extent, though it is not possible to reach the general endometrial level. So, in terms of clinical aspects, the expression of HOXA10mRNA by the endometrial cells decreases significantly when blocking the hydrosalpinx. ConclusionsAmong hydrosalpinx patients, one of the major mechanisms that damage the endometrium was found to be the abnormal expression of HOXA10 followed by IGFBP1 and avβ3, its downstream genes. This further results in the implantation of the embryo as well. Though it is possible to gradually repair the damage after the removal of hydrosalpinx, the recovery is a time-consuming process.

Safety and immunogenicity of TG4050: A personalized cancer vaccine in head and neck carcinoma.

6082 Background: Despite adjuvant therapy, over 50% of surgically treated head and neck squamous cell carcinoma (HNSCC) patients (pts) experience a recurrence of disease. Systemic stimulation of cellular immunity against tumor mutations using a viral vaccine may be an ideal modality to clear residual cancer cells. For this purpose, we developed a pipeline for the design of TG4050, a personalized cancer vaccine (PCV) using a Modified Vaccinia Ankara (MVA) viral vector. We report here preliminary safety and immunogenicity data from a phase I TG4050 study. Methods: Surgically resected stage III or IV, HPV negative HNSCC pts were enrolled in the study. pts must have achieved clinical remission after adjuvant chemoradiotherapy. A PCV for each pt was manufactured with up to 30 neoantigens identified using a state-of-the-art machine learning algorithm, from next generation sequencing (NGS) data. Pts randomized to arm A received the PCV after completion of primary treatment. Pts randomized to arm B received the PCV in the event of relapse, in conjunction with second line therapy. The PCV schedule consisted of an induction period of 6 weekly administrations, followed by booster doses once every 3 weeks for up to one year. Immune cells were collected by leukapheresis at baseline and at day 64. Primary endpoint was safety. Secondary endpoints included feasibility, disease free survival and immune response as assessed by ex-vivo IFNg-ELISPOT. Results: At the time of data cut-off, a total of 31 pts were randomized, 15 in arm A and 16 in arm B. A vaccine was successfully designed for all randomized pts. Pts had no evidence of disease at baseline either at the clinical or molecular level, as assessed by ctDNA assessment. All adverse events (AEs) were mild to moderate and most were injection site reactions. Median follow-up was 9.2 months in arm A vs 7.6 months in arm B. None of the pts in arm A experienced relapse vs. 2 in the arm B. Immune monitoring demonstrated priming of a polyepitopic T cell response against the PCV in 100% of pts in arm A, among pts evaluated to date, with a mean of 9 responses per pt (6-19). Responses were observed regardless of HLA genotype, and without cross-reactivity to the wildtype antigen. Baseline tumor analyses revealed challenging genomic and immune profiles such as low TMB (avg of 3.06 ± 0.86 Mut/Mb), a majority of immune-desert tumors, and a low expression of important immune related factors including PD-L1 (16 pts out of 17 had a negative to moderate PD-L1 expression). Conclusions: Our preliminary data demonstrate that TG4050 is safe, well tolerated, and capable of inducing T cell responses in cold tumors. In summary viral based, PCVs designed to induce tumor-specific neoantigen may be associated with a safe tolerance and an improved outcome in HNSCC pts. Clinical trial information: NCT04183166 .

Study on the therapeutic effect and mechanism of Tangningtongluo Tablet on diabetic mice

AimsTo investigate the therapeutic effects of Tangningtongluo Tablet on diabetic mice and its mechanism. This study was established the scientific basis for the clinical application of Tangningtongluo Tablet in the treatment of diabetes mellitus and provided data supporting the transformation of Tangningtongluo Tablet from an in-hospital preparation to a new Chinese medicine. MethodsIn this study, a diabetic mouse model was established by high-glucose and high-fat diet feeding in combination with STZ injection for 4 weeks. Glucose metabolism, lipid metabolism, liver histomorphological changes and liver function related indexes were detected, pancreatic histomorphological changes and insulin resistance related indexes were observed, and the expression of pathway related proteins and inflammatory factors were examined. ResultsGlycemia and glycated hemoglobin were reduced in diabetic mice after the treatment of Tangningtongluo Tablet, and glucose tolerance and lipid results were modified. The insulin resistance status of the mice was diminished and tissue damage to the pancreas and liver was repaired. Expression of ERS/NF-κB related pathway proteins was reduced in liver tissues, and inflammatory factors such as TNF-α, IL-6 and IL-1β were reduced in serum. ConclusionsTangningtongluo Tablet could reduce blood glucose in diabetic mice, regulate the disorder of lipid metabolism, enhance insulin sensitivity, improve insulin resistance, repair pancreatic tissue damage and protect mouse liver in diabetic mice. The mechanism of action might be related to the regulation of ERS/NF-κB signaling pathway and the reduction of TNF-α, IL-6 and IL-1β production.

HMGA2 knockdown alleviates the progression of nonalcoholic fatty liver disease (NAFLD) by downregulating SNAI2 expression

Non-alcoholic fatty liver disease (NAFLD) is a complex disease that is considered as the next major health epidemic with alarmingly increasing global prevalence. To explore the pathogenesis of NAFLD, data from GSE118892 were analyzed. High mobility group AT-hook 2 (HMGA2), a member of the high mobility group family, is declined in liver tissues of NAFLD rats. However, its role in NAFLD remains unknown. This study attempted to identify the multiple roles of HMGA2 in NAFLD process. NAFLD was induced in rats using a high-fat diet (HFD). In vivo, HMGA2 knockdown using adenovirus system attenuated liver injury and liver lipid deposition, accompanied by decreased NAFLD score, increased liver function, and decreased CD36 and FAS, indicating the deceleration of NAFLD progression. Moreover, HMGA2 knockdown restrained liver inflammation by decreasing the expression of related inflammatory factors. Importantly, HMGA2 knockdown attenuated liver fibrosis via downregulating the expression of fibrous proteins, and inhibiting the activation of TGF-β1/SMAD signaling pathway. In vitro, HMGA2 knockdown relieved palmitic acid (PA)-induced hepatocyte injury and attenuated TGF-β1-induced liver fibrosis, consistent with in vivo findings. Strikingly, HMGA2 activated the transcription of SNAI2, which was evidenced by the dual luciferase assays. Moreover, HMGA2 knockdown largely downregulated SNAI2 levels. Indeed, SNAI2 overexpression effectively blocked the inhibitory effect of HMGA2 knockdown on NAFLD. Totally, our findings reveal that HMGA2 knockdown alleviates the progression of NAFLD by directly regulating the transcription of SNAI2. HMGA2 inhibition may emerge as a potential therapeutic target for NAFLD.

STA-21, a small molecule STAT3 inhibitor, ameliorates experimental autoimmune encephalomyelitis by altering Th-17/Treg balance.

Numerous studies have demonstrated the role of T helper (Th) 17 and T regulatory (reg) cells and pro-inflammatory and anti-inflammatory cytokines related to these cells in the pathogenesis of MS and its animal model, experimental autoimmune encephalomyelitis (EAE). STAT3 is one of the downstream signaling proteins of IL-23, IL-6, and IL-21 that are required for Th17 cells differentiation. STA-21 is a STAT3 inhibitor that functions by inhibiting STAT3 dimerization and binding to DNA impairing the expression of STAT3 target genes including, RORγt, IL-21 and IL-23R that are also required for Th17 cell differentiation. In this study, we evaluated the effect of STA-21 on EAE Model and investigated how this small molecule can change Th17/Treg balance leading to amelioration of disease. After EAE induction and treatment with STA-21, its effects were assessed. Major assays were H&E and LFB staining, Flow cytometric analysis, Reverse transcription-PCR (RT-PCR), and ELISA. STA-21 ameliorated the EAE severity and decreased the EAE inflammation and demyelination. It also decreased STAT3 phosphorylation, the proportion of Th17 cells and the protein level of IL-17. In contrast, the balance of Tregs and the level of anti-inflammatory cytokine, IL-10 increased in STA-21-treated mice. Moreover, STA-21 significantly decreased the expression of Th17 related transcription factors, RORɣt and IL-23R while FOXP3 expression associated with Treg differentiation was increased. This study showed that STA-21 has therapeutic effects in EAE by reducing inflammation and shifting inflammatory immune responses to anti-inflammatory and can be used as a suitable treatment strategy for the treatment of EAE. The effectiveness of inhibiting or strengthening the functional cells of the immune system by these small molecules in terms of easy to access, simple construction and inexpensive expansion make them as a suitable tool for the treatment of inflammatory and autoimmune diseases.

Enoxaparin sodium bone cement plays an anti-inflammatory immunomodulatory role by inducing the polarization of M2 macrophages

ObjectiveThe implantation of PMMA bone cement results in an immune response and the release of PMMA bone cement particles causes an inflammatory cascade. Our study discovered that ES-PMMA bone cement can induce M2 polarization of macrophages, which has an anti-inflammatory immunomodulatory effect. We also delved into the molecular mechanisms that underlie this process.MethodsIn this study, we designed and prepared samples of bone cement. These included PMMA bone cement samples and ES-PMMA bone cement samples, which were implanted into the back muscles of rats. At 3, 7, and 14 days after the operation, we removed the bone cement and a small amount of surrounding tissue. We then performed immunohistochemistry and immunofluorescence to observe the polarization of macrophages and the expression of related inflammatory factors in the surrounding tissues. The RAW264.7 cells were exposed to lipopolysaccharide (LPS) for 24 h to establish the macrophage inflammation model. Then, each group was treated with enoxaparin sodium medium, PMMA bone cement extract medium, and ES-PMMA bone cement extract medium, respectively, and cultured for another 24 h. We collected cells from each group and used flow cytometry to detect the expressions of CD86 and CD206 in macrophages. Additionally, we performed RT-qPCR to determine the mRNA levels of three markers of M1 macrophages (TNF-α, IL-6, iNOS) and two M2 macrophage markers (Arg-1, IL-10). Furthermore, we analyzed the expression of TLR4, p-NF-κB p65, and NF-κB p65 through Western blotting.ResultsThe immunofluorescence results indicate that the ES-PMMA group exhibited an upregulation of CD206, an M2 marker, and a downregulation of CD86, an M1 marker, in comparison to the PMMA group. Additionally, the immunohistochemistry results revealed that the levels of IL-6 and TNF-α expression were lower in the ES-PMMA group than in the PMMA group, while the expression level of IL-10 was higher in the ES-PMMA group. Flow cytometry and RT-qPCR analyses revealed that the expression of M1-type macrophage marker CD86 was significantly elevated in the LPS group compared to the NC group. Additionally, M1-type macrophage-related cytokines TNF-α, IL-6, and iNOS were also found to be increased. However, in the LPS + ES group, the expression levels of CD86, TNF-α, IL-6, and iNOS were decreased, while the expression of M2-type macrophage markers CD206 and M2-type macrophage-related cytokines (IL-10, Arg-1) were increased compared to the LPS group. In comparison to the LPS + PMMA group, the LPS + ES-PMMA group demonstrated a down-regulation of CD86, TNF-α, IL-6, and iNOS expression levels, while increasing the expression levels of CD206, IL-10, and Arg-1. Western blotting results revealed a significant decrease in TLR4/GAPDH and p-NF-κB p65/NF-κB p65 in the LPS + ES group when compared to the LPS group. Additionally, the LPS + ES-PMMA group exhibited a decrease in TLR4/GAPDH and p-NF-κB p65/NF-κB p65 levels when compared to the LPS + PMMA group.ConclusionES-PMMA bone cement is more effective than PMMA bone cement in down-regulating the expression of the TLR4/NF-κB signaling pathway. Additionally, it induces macrophages to polarize towards the M2 phenotype, making it a crucial player in anti-inflammatory immune regulation.Graphical