VEGF mRNA Expression Research Articles

Angiogenic genes and proteins are differentially expressed in left and right ventricular tissue in perinatal sheep hearts

Birth places a great stress on the fetal heart due to increased cardiac workload, following a disconnection from the low-resistance placenta and closure of fetal shunts. The mechanisms that regulate myocardial microvascular growth and development during the perinatal period are largely unknown. We hypothesized that birth stimulates an increase in angiogenic gene and protein expression in a ventricular dependent manner to reflect the changing roles for the left and right ventricles (LV, RV). Fetal (135 d gestation, dGA; term=147 dGA) and neonatal (1 and 5 postnatal days, PD) sheep LV and RV were snap frozen. Gene expression of key angiogenic regulators ( VEGFA, VEGFR1, VEGFR2, ANGPT1, ANGPT2, PGC1A), endothelial cell growth ( DDL4, NOTCH4, SPP1, PECAM1, NCL), nitric oxide synthesis ( NOS2, NOS3), and extracellular matrix (ECM) remodeling ( MMP2, MMP9, THBS1) were quantified using real-time qPCR (n=12/age group). Protein expression of VEGFA and VEGF-R2 were measured by Western blot (n=5/age group). Data were analyzed by two-way ANOVA with Tukey’s post-hoc test to determine effects of age, ventricle, and interaction. Significance was set at P<0.05. At 135 dGA, all angiogenic, nitric oxide synthesis, ECM, DDL4, SPP1, and PECAM1 mRNA expression levels were higher in the RV vs. LV. At PD1, there were no differences between LV and RV. At PD5, VEGFA, VEGFR1, ANGPT1, ANGPT2, PGC1A, DDL4, SPP1, PECAM1, NCL, MMP2, and THBS1 mRNA expression levels were again higher in the RV vs. LV. At 135 dGA, VEGFA and VEGF-R2 protein expression levels were 76% and 72% lower, respectively, in the RV vs. LV. Within each ventricle, age related changes to mRNA expression patterns were unique. In the LV, mRNA expression of NOTCH4 and NCL increased stepwise from 135 dGA to PD5; however, in the RV, both were upregulated at PD1 but returned to fetal levels by PD5. In contrast, all angiogenic, ECM, DDL4, SPP1, PECAM1, and NOS2 genes were downregulated in the RV at PD1 but all returned to fetal levels by PD5. In the LV, VEGF-R2 protein expression was 56% and 73% lower at PD 1 and 5, respectively, vs. 135 dGA. In summary, gene and protein expression patterns involved in angiogenesis and vascular remodeling change over the perinatal period and are differentially expressed in the ventricles. Greater angiogenic gene expression levels in the RV vs. the LV at 135 dGA might be due to the RV serving as the main systemic pump in utero, thus requiring relatively increased microcirculation. However, reduced VEGFA and VEGF-R2 protein levels at 135 dGA contradict this hypothesis; histological studies to determine vascularity are ongoing. Likewise, the subsequent downregulation of angiogenic genes in the RV at PD1 might be due to the RV’s sudden decrease in cardiac output as the LV becomes the main pump. The underlying reasons for why LV angiogenic genes are not significantly upregulated in the early postnatal period, given its new role as the main systemic pump, are yet to be elucidated. NIH R21-HL152112 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Anti-scarring effects of conbercept on human Tenon’s fibroblasts: comparisons with bevacizumab

BackgroundSafely inhibiting the formation of scar in the glaucoma filtration surgery (GFS) has always been an issue for clinical glaucoma doctors. Anti-vascular endothelial growth factor (VEGF) agents can reduce angiogenesis, and anti-placental growth factor (PIGF) agents can affect reactive gliosis. However, the effect of conbercept, which can bind to both VEGF and PIGF, on human Tenon’s fibroblasts (HTFs) is unknown.MethodsHTFs were cultured in vitro and treated with conbercept or bevacizumab (BVZ). No drug was added to the control group. The effects of drugs on cell proliferation were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and the collagen type I alpha1(Col1A1) mRNA expression level was measured using quantitative polymerase chain reaction (qPCR). HTF cell migration after drug interventions was evaluated using the scratch wound assay along with the measurement of the expression levels of VEGF and PIGF in human umbilical vein endothelial cells (HUVECs) using enzyme-linked immunosorbent assay, as well as the detection of the VEGF(R) mRNA expression level in HTFs using qPCR.ResultsAfter the addition of conbercept (0.01, 0.1, and 1 mg/mL) to the cultured HTFs or HUVECs, no significant cytotoxicity was observed compared with the control group, while the cytotoxicity of 2.5 mg/mL BVZ on HTFs was obvious. Conbercept significantly inhibited HTF cell migration and Col1A1 mRNA expression level in HTFs. It was superior to BVZ in inhibiting HTF migration. After the intervention with conbercept, the expression level of PIGF and VEGF in HUVECs significantly decreased; and the inhibitory effect of conbercept on the expression level of VEGF in HUVECs was weaker than that of BVZ. Conbercept was more advantageous than BVZ in inhibiting the expression level of VEGFR-1 mRNA in HTFs. However, its effect in terms of inhibiting the expression level of VEGFR-2 mRNA in HTFs was weaker than that of BVZ.ConclusionThe results suggested the low cytotoxicity and significant anti-scarring effect of conbercept in HTF with significant anti-PIGF and inferior anti-VEGF effects compared with BVZ, thus providing a better understanding of the role of conbercept in the GFS wound healing process.

Vascular characterization and morphogenesis in porcine placenta at day 40 of gestation.

In porcine placenta, abnormal development of the placental vasculature leads to placental insufficiency. The aim of this study was to determine the mRNA expression of angiogenic growth factors and to determine the vascular characteristics in placenta at day 40 of pig gestation. Samples were collected from maternal-chorioallantoic interface (n = 21) for the measurement of mRNA expression of VEGFA, ANGPT1, ANGPT2, FGF2 and its receptors KDR, TEK, FGFR1IIIc, FGFR2IIIb respectively, and for immunohistochemistry analysis of CD31 and VEGFA. Immunohistochemical analysis of CD31 and VEGFA, morphometric measurement of blood vessels, high resolution light microscopy and transmission electron microscopy were performed. Capillary area density, number of blood vessels and capillary area were significantly higher on the maternal side than on the fetal side (P<0.05). The ultrastructural finding of blood vessels demonstrates close contact with the trophoblastic epithelium. The relative mRNA expression of VEGFA and its receptor KDR were higher compared with the other angiogenic genes. In conclusion, a high mRNA expression of VEGFA and its receptor KDR added to the immunohistochemical results suggest a potential role of these genes in this pathway associated with an increase in the density of the capillary area on the maternal side and a reduction in the hemotrophic diffusion distance at the interface for nutrient exchange.

Deciphering the mechanisms of Yinlan Tiaozhi capsule in treating hyperlipidemia by combining network pharmacology, molecular docking and experimental verification

Yinlan Tiaozhi capsule (YLTZC) has been widely used to treat hyperlipidemia (HLP). However, its material basis and underlying pharmacological effects remain unclean. The current study aimed to explore the mechanisms involved in the treatment of YLTZC on HLP based on network pharmacology, molecular docking, and experimental verification. Firstly, UPLC-Q-TOF–MS/MS was used to comprehensively analyze and identify the chemical constituents in YLTZC. A total of 66 compounds, mainly including flavonoids, saponins, coumarins, lactones, organic acids, and limonin were characterized and classified. Simultaneously, the mass fragmentation pattern of different types of representative compounds was further explored. By network pharmacology analysis, naringenin and ferulic acid may be the core constituents. The 52 potential targets of YLTZC, including ALB, IL-6, TNF, and VEGFA, were considered potential therapeutic targets. Molecular docking results showed that the core active constituents of YLTZC (naringenin and ferulic acid) have a strong affinity with the core targets of HLP. Lastly, animal experiments confirmed that naringenin and ferulic acid significantly upregulated the mRNA expression of ALB and downregulated the mRNA expression of IL-6, TNF, and VEGFA. In sum, the constituents of YLTZC, such as naringenin and ferulic acid, might treat HLP by regulating the mechanism of angiogenesis and inhibiting inflammatory responses. Furthermore, our data fills the gap in the material basis of YLTZC.

Bioinformatics analysis of potential key ferroptosis-related genes involved in tubulointerstitial injury in patients with diabetic nephropathy

Diabetic nephropathy (DN) is the primary complication of diabetes mellitus. Ferroptosis is a form of cell death that plays an important role in DN tubulointerstitial injury, but the specific molecular mechanism remains unclear. Here, we downloaded the DN tubulointerstitial datasets GSE104954 and GSE30529 from the Gene Expression Omnibus database. We examined the differentially expressed genes (DEGs) between DN patients and healthy controls, and 36 ferroptosis-related DEGs were selected. Pathway-enrichment analyses showed that many of these genes are involved in metabolic pathways, phosphoinositide 3-kinase/Akt signaling, and hypoxia-inducible factor-1 signaling. Ten of the 36 ferroptosis-related DEGs (CD44, PTEN, CDKN1A, DPP4, DUSP1, CYBB, DDIT3, ALOX5, VEGFA, and NCF2) were identified as key genes. Expression patterns for six of these (CD44, PTEN, DDIT3, ALOX5, VEGFA, and NCF2) were validated in the GSE30529 dataset. Nephroseq data indicated that the mRNA expression levels of CD44, PTEN, ALOX5, and NCF2 were negatively correlated with the glomerular filtration rate (GFR), while VEGFA and DDIT3 mRNA expression levels were positively correlated with GFR. Immune infiltration analysis demonstrated altered immunity in DN patients. Real-time quantitative PCR (qPCR) analysis showed that ALOX5, PTEN, and NCF2 mRNA levels were significantly upregulated in high-glucose-treated human proximal tubular (HK-2) cells, while DDIT3 and VEGFA mRNA levels were significantly downregulated. Immunohistochemistry analysis of human renal biopsies showed positive staining for ALOX5 and NCF2 protein in DN samples but not the controls. These key genes may be involved in the molecular mechanisms underlying ferroptosis in patients with DN, potentially through specific metabolic pathways and immune/inflammatory mechanisms.

Differential Impact of Intermittent vs. Sustained Hypoxia on HIF-1, VEGF and Proliferation of HepG2 Cells.

Obstructive sleep apnea (OSA) is an emerging risk factor for cancer occurrence and progression, mainly mediated by intermittent hypoxia (IH). Systemic IH, a main landmark of OSA, and local sustained hypoxia (SH), a classical feature at the core of tumors, may act separately or synergistically on tumor cells. Our aim was to compare the respective consequences of intermittent and sustained hypoxia on HIF-1, endothelin-1 and VEGF expression and on cell proliferation and migration in HepG2 liver tumor cells. Wound healing, spheroid expansion, proliferation and migration were evaluated in HepG2 cells following IH or SH exposure. The HIF-1α, endothelin-1 and VEGF protein levels and/or mRNA expression were assessed, as were the effects of HIF-1 (acriflavine), endothelin-1 (macitentan) and VEGF (pazopanib) inhibition. Both SH and IH stimulated wound healing, spheroid expansion and proliferation of HepG2 cells. HIF-1 and VEGF, but not endothelin-1, expression increased with IH exposure but not with SH exposure. Acriflavine prevented the effects of both IH and SH, and pazopanib blocked those of IH but not those of SH. Macitentan had no impact. Thus, IH and SH stimulate hepatic cancer cell proliferation via distinct signaling pathways that may act synergistically in OSA patients with cancer, leading to enhanced tumor progression.

Effect of topical motesanib in experimental corneal neovascularization model.

This study aimed to compare the efficacy of topical bevacizumab and motesanib in an experimental corneal neovascularization model, and find the most effective motesanib dose. In experiments, 42 Wistar Albino rats were randomly divided into six groups (n = 7). Corneal cauterization was applied to all groups except the group 1. Group 1did not receive any treatment. Topical dimethylsulfoxide was applied toshamgroupthree times a day(tid). Topical bevacizumab drops (5mg/ml) were applied to Group 3 tid. Topical motesanib drops with a dose of 2.5, 5, and 7.5mg/ml were respectively applied in Groups 4, 5, and 6 tid. On the 8th day, corneal photographs of all rats were taken under general anesthesia, and the percentage of corneal neovascular area was calculated. VEGF-A mRNA, VEGFR-2 mRNA, miRNA-21, miRNA-27a, miRNA-31, miRNA-126, miRNA-184, and miRNA-204 were evaluated by the qRT-PCR method in corneas taken after decapitation. The percentage of corneal neovascularization areas and VEGF-A mRNA expression levels were decreased in all treatment groups compared to group 2 (p < 0.05). VEGFR-2 mRNA levels were found to be statistically significantly decreased in groups 4 and 6 compared to group 2 (p < 0.05).Statistically significant changes were detected in the expression levels of only miRNA-126 among all miRNAs. Motesanib with a dose of 7.5mg/ml statistically significantly suppressed the VEGFR-2 mRNA level compared with other treatment doses and may be more effective than bevacizumab. Further, miRNA-126 can be used as a proangiogenic marker.

Loss of Histone Methyltransferase KMT2D Attenuates Angiogenesis in the Ischemic Heart by Inhibiting the Transcriptional Activation of VEGF-A

Angiogenesis occurred after myocardial infarction (MI) protects heart failure (HF). The aim of our study was to explore function of histone methyltransferase KMT2D (MLL4, mixed-lineage leukemia 4) in angiogenesis post-MI. Western blotting showed that KMT2D protein expression was elevated in MI mouse myocardial. Cardiomyocyte-specific Kmt2d-knockout (Kmt2d-cKO) mice were generated, and echocardiography and immunofluorescence staining detected significantly attenuated cardiac function and insufficient angiogenesis following MI in Kmt2d-cKO mice. Cross-talk assay suggested that Kmt2d-KO H9c2-derived conditioned medium attenuates EA.hy926 EC function. ELISA further identified that VEGF-A released from Kmt2d-KO H9c2 was significantly reduced. CUT&Tag and RT-qPCR revealed that KMT2D deficiency reduced Vegf-a mRNA expression and enrichment of H3K4me1 on the Vegf-a promoter. Moreover, KMT2D silencing in ECs also suppressed endothelial function. Our study indicates that KMT2D depletion in both cardiomyocytes and ECs attenuates angiogenesis and that loss of KMT2D exacerbates heart failure after MI in mice.

The Tpl2-MEK pathway plays a critical role in spheroid-cultured endometriotic stromal cells.

Endometriosis is a proliferative disease characterized by cytokine-induced inflammation. The objective of this study was to assess cell growth and PGE2 production induced by TNF-α in endometriotic stromal cells (ESCs) in spheroid cell culture and to identify the signaling pathway involved with a view to finding new therapeutic targets for endometriosis. Tissue samples were collected from patients with and without endometriosis. ESCs were isolated from ovarian endometrioma (OE). Gene expression was evaluated by real-time PCR and DNA microarray analysis, the proliferative effect on ESCs by WST-8 assay, and PGE2 production by ELISA. Protein phosphorylation was detected using western blotting. COX-2, aromatase and VEGFA mRNA expression and PGE2 production were significantly elevated in spheroid cell cultures compared to monolayer cell cultures. TNF-α receptor (TNFR) 1 and TNFR2 mRNA was also significantly increased. TNF-α promoted the proliferation and PGE2 production of ESCs in spheroid cell cultures significantly more than in monolayer cell cultures. TNF-α increased the expression of several genes related to the pathophysiology of endometriosis in spheroid ESCs. DNA microarray analysis revealed that the Tpl2 gene, which codes for a MAPK upstream of MEK, was upregulated in OE and endometrium with endometriosis compared to normal endometrium. TNF-α increased the phosphorylation and expression of Tpl2 and MEK, and Tpl2 and MEK inhibitors inhibited TNF-α-induced proliferation and PGE2 production in spheroid ESCs. The Tpl2-MEK signaling pathway may play a critical role in the cell growth and PGE2 production induced by TNF-α in spheroid ESCs.

Helicobacter pylori infection induces gastric precancerous lesions and persistent expression of Angpt2, Vegf-A and Tnf-A in a mouse model.

Helicobacter pylori colonizes the gastric mucosa and induces chronic inflammation. Using a mouse model of H. pylori-induced gastritis, we evaluated the mRNA and protein expression levels of proinflammatory and proangiogenic factors, as well as the histopathological changes in gastric mucosa in response to infection. Five- to six-week-old female C57BL/6N mice were challenged with H. pylori SS1 strain. Animals were euthanized after 5-, 10-, 20-, 30-, 40- and 50-weeks post infection. mRNA and protein expression of Angpt1, Angpt2, VegfA, Tnf-α, bacterial colonization, inflammatory response and gastric lesions were evaluated. A robust bacterial colonization was observed in 30 to 50 weeks-infected mice, which was accompanied by immune cell infiltration in the gastric mucosa. Compared to non-infected animals, H. pylori-colonized animals showed an upregulation in the expression of Tnf-A, Angpt2 and VegfA at the mRNA and protein levels. In contrast, Angpt1 mRNA and protein expression was downregulated in H. pylori-colonized mice. Our data show that H. pylori infection induces the expression of Angpt2, Tnf-A and Vegf-A in murine gastric epithelium. This may contribute to the pathogenesis of H. pylori-associated gastritis, however the significance of this should be further addressed.

Combination of Radix Astragali and Safflower Promotes Angiogenesis in Rats with Ischemic Stroke via Silencing PTGS2.

Promotion of angiogenesis and restoration of the blood flow in the ischemic penumbra is an effective treatment for patients with ischemic stroke (IS). Radix astragali-safflower (AS), a classic herbal pair for accelerating blood circulation and dispersing blood stasis, has been used for thousands of years to treat patients with IS in China. Even so, the mechanism of the treatment of IS by AS is still undecipherable. In the current study, network pharmacology was firstly employed to unveil the mechanism of AS in treating IS, which showed that AS might promote angiogenesis associated with PTGS2 silence. Middle cerebral artery occlusion/reperfusion (MCAO/R) model rats were then used as the experimental animals to verify the prediction result. The experimental results revealed that treatment with AS improved the cerebral infarct volume, neurological damage, and cerebral histopathological damage; inhibited cell apoptosis; increased the contents of PDGF-BB, EPO, and TGF-β1; and reduced the levels of PF4, Ang-2, and TIMP-1 in serum. Immunohistochemical staining demonstrated that the expression of PTGS2 was dramatically increased in the hippocampus and cerebral cortex of rats with MCAO/R, and this trend was reversed by the treatment of AS. Immunofluorescent staining expressed that AS reversed the down-regulation of VEGF and further promoted the expression of CD31, which indicated that AS promoted angiogenesis in MCAO/R rats. The abnormal protein or mRNA expression of PTGS2, PGI2, bFGF, TSP-1, and VEGF in the penumbra were transposed by AS or Celecoxib (an inhibitor of PTGS2). In conclusion, the protective mechanism of AS for IS promoted angiogenesis and was involved with PTGS2 silence.

Exploring the mechanism by which aqueous Gynura divaricata inhibits diabetic foot based on network pharmacology, molecular docking and experimental verification

BackgroundTo predict and validate the potential mechanism by which Gynura divaricata (GD) functions in the treatment of diabetic foot (DF).MethodsThe main chemical constituents of GD were identified by reviewing the literature, the traditional Chinese medicine database platform (TCMIP) and the BATMAN-TCM platform. DF disease targets were identified with the GeneCards database, and the compound-target network was constructed by using the intersection of drugs and disease. The STRING platform was used to construct the protein–protein interaction (PPI) network, and Cytoscape 3.7.2 software was used to visualize the results. Moreover, the Metascape database was used for Gene Ontology (GO) enrichment analyses and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Molecular docking of the active ingredients of GD and core protein targets of DF was performed using AutoDock software. Finally, the predicted results were preliminarily verified with experiments.ResultsA total of 140 potential targets of GD were identified and associated with DF. According to the PPI network analysis, GD accelerated DF wound healing, and the mechanism may be related to proteins such as AKT1, TP53, IL6, CASP3, TNF, and VEGFA. GO and KEGG enrichment analyses indicated that GD may play a role in the treatment of diabetic foot by affecting various signaling pathways. Molecular docking results showed that the proteins AKT1, TP53, IL6, CASP3, TNF, and VEGFA were closely associated with the components of GD. The animal experiments showed that GD reduced the levels of IL-6 and TNF-α and increased the mRNA and protein expression of VEGFA in rats with DF.ConclusionsGD regulates multiple targets and multiple pathways to promote wound healing in DF.

Expression of PKM2 in wound keratinocytes is coupled to angiogenesis during skin repair in vivo and in HaCaT keratinocytes in vitro

An injured skin is rapidly restored in a manner of wound healing. We have previously shown that intact insulin signaling and glucose uptake are fundamental to proper wound closure. Consequently, under exacerbated inflammation, compromised insulin action and glucose uptake lead to impaired healing. However, in spite of the increased attention to cell metabolism during tissue regeneration, metabolic mediators that govern cellular and physiological processes throughout skin repair remained largely elusive. Through assessment of mRNA using real-time PCR and protein blot analysis, we report that healing of cutaneous wounds comprise a boosted expression of genes involved in glycolysis, oxidative phosphorylation, pentose phosphate shunt, and glutamine anaplerosis. We further focused on the functional role of pyruvate kinase M (PKM) isoenzymes that catalyze the final and rate-limiting step of glycolysis. Whereas the expression of the metabolic constitutively active Pkm1 isozyme remained almost unchanged, Pkm2 is augmented during the inflammatory phase of healing. The immunohistochemistry and RNA in situ hybridization analysis showed a confined Pkm2 expression to keratinocytes of the hyperproliferative epithelium and, to a lesser extent, infiltrating neutrophils and monocytes as well as later on in macrophages. Notably, the expression of Pkm2 in keratinocytes facing the wound bed side colocalized with VEGF expression. The in vitro knockdown of PKM2 in HaCaT keratinocytes using small interfering (si) RNA confirmed an acute role for PKM2 in facilitating the complete induction of VEGF mRNA and protein expression in keratinocytes; this function is mainly HIF-1α independent.Key messages• Wound healing involves activation of glycolysis, oxidative phosphorylation, pentos-phosphate shunt, and replenishment of tri-carboxylic acid (TCA) cycle through glutamine anaplerosis.• The pyruvate kinase M2 (PKM2) isoform is upregulated during the inflammatory phase of cutaneous healing, mainly in keratinocytes of hyperproliferative epithelia.• In vivo, the expression of VEGF in wound keratinocytes is colocalized with PKM2.• PKM2 is required for full induction of VEGF in HaCaT keratinocytes in vitro.

The effect of thymoquinone and propranolol combination on epidermoid laryngeal carcinoma cell.

We aimed to evaluate the effects of thymoquinone and propranolol on Hep-2 cells representing laryngeal Ca cell type in comparison with cisplatin. We also evaluated their combined effects. Apoptotic effects were directly analyzed via mitochondrial membrane potential and caspase-3 assays. In addition, effects on apoptosis and cell cycle via Bcl-2, Bax, P53, and Cyclin D1 mRNA expressions and effects on angiogenesis via VEGFA mRNA expression were evaluated by RT-qPCR. According to our results, it was determined that the anticancer effects of thymoquinone on Hep-2 cells were higher than propranolol. Our JC-1 and caspase-3 results showed an effect close to cisplatin, especially for 50µM thymoquinone. Significant differences were also obtained in Bcl-2, Bax, P53, and cyclin D1 results for similar concentrations compared to the control. No effect of thymoquinone was seen for VEGFA. Propranolol alone had no significant effect on JC-1 and Caspase-3. Propranolol had an effect on Bcl-2, Bax mRNA expressions compared to the control, only at 250µM concentration. Propranolol and its combinations increased VEGFA mRNA expression-like cisplatin. Thymoquinone induced apoptosis and blocked the cell cycle in Hep-2 cells. The effects of propranolol, which was reported to have an antiangiogenesis effect in some studies, on apoptosis and cell cycle were limited except at high concentrations. For this cell line, why propranolol causes an increase in VEGFA expression should be evaluated extensively. Thymoquinone shows promise for cancer therapy, but studies need to be designed in vivo to evaluate the effects more reliably.

Molecular docking, network pharmacology and experimental verification to explore the mechanism of Wulongzhiyangwan in the treatment of pruritus

Wulongzhiyangwan (WLZYW) is a Chinese prescription medicine for the treatment of pruritus, but its mechanism has not been clarified. The purpose of this study was to explore the mechanism of WLZYW in pruritus through network pharmacology analysis and experimental validation. The active components and corresponding targets of WLZYW were obtained from the Traditional Chinese Medicine Systematic Pharmacology (TCMSP) database. Pruritus-related targets were obtained from the GeneCards, TTD (Therapeutic Target Database), and DrugBank databases. The key compounds, core targets, main biological processes and signaling pathways related to WLZYW were identified by constructing and analyzing related networks. The binding affinity between WLZYW components and core targets was validated by AutoDock Vina software. In this study, RBL-2H3 cells were used to construct a degranulation model to simulate histamine-dependent pruritus. 10 chemical constituents, 235 targets and 3606 pruritus-related targets of WLZYW were obtained. Subsequently, 26 core targets were identified through analysis, VEGFA and AKT1 were the main candidates. A pathway enrichment analysis showed that overlapping targets were significantly enriched in the PI3K/AKT signaling pathway. A molecular docking analysis revealed tight binding of VEGF to three core compounds, kaempferol, luteolin and quercetin. Experiments showed that WZLYW inhibited mast cell degranulation, regulated VEGFa mRNA and protein expression levels by inhibiting PI3K/AKT and ERK1/2 signaling pathway activation. The mechanism of WZLYW in pruritus may be regulating VEGFa expression. Network pharmacology assays suggested that WLZYW downregulates VEGFa expression by regulating the PI3K/AKT and ERK1/2 signaling pathways in pruritis treatment.

Cannabidiol Rescues TNF-α-Inhibited Proliferation, Migration, and Osteogenic/Odontogenic Differentiation of Dental Pulp Stem Cells.

Strategies to promote dental pulp stem cells (DPSCs) functions including proliferation, migration, pro-angiogenic effects, and odontogenic/osteogenic differentiation are in urgent need to restore pulpitis-damaged dentin/pulp regeneration and DPSCs-based bone tissue engineering applications. Cannabidiol (CBD), an active component of Cannabis sativa has shown anti-inflammation, chemotactic, anti-microbial, and tissue regenerative potentials. Based on these facts, this study aimed to analyze the effect of CBD on DPSCs proliferation, migration, and osteogenic/odontogenic differentiation in basal and inflammatory conditions. Highly pure DPSCs with characteristics of mesenchymal stem cells (MSCs) were successfully isolated, as indicated by the results of flowcytometry and multi-lineage (osteogenic, adipogenic, and chondrogenic) differentiation potentials. Among the concentration tested (0.1-12.5 µM), CBD (2.5 μM) showed the highest anabolic effect on the proliferation and osteogenic/odontogenic differentiation of DPSCs. Pro-angiogenic growth factor VEGF mRNA expression was robustly higher in CBD-treated DPSCs. CBD also prompted the migration of DPSCs and CBD receptor CB1 and CB2 expression in DPSCs. TNF-α inhibited the viability, migration, and osteogenic/odontogenic differentiation of DPSCs and CBD reversed these effects. CBD alleviated the TNF-α-upregulated expression of pro-inflammatory cytokines TNF-α, interleukin (IL)-1β, and IL-6 in DPSCs. In conclusion, our results indicate the possible application of CBD on DPSCs-based dentin/pulp and bone regeneration.

Modulation of Hypoxia-Inducible Factors and Vascular Endothelial Growth Factor Expressions by Superfood Camu-Camu (Myrciaria dubia) Treatment in ARPE-19 and Fetal Human RPE Cells.

Anti-vascular endothelial growth factor (anti-VEGF) therapy via intravitreal injection is an effective treatment for patients with abnormal ocular neovascularization, such as age-related macular degeneration (AMD) and diabetic macular edema (DME). However, prolonged and frequent anti-VEGF treatment is associated with a risk of local and systemic adverse events, including geographic atrophy, cerebrovascular disease, and death. Furthermore, some patients do not adequately respond to anti-VEGF therapy. Hypoxia-inducible factor (HIF) is a transcription factor that controls the expression of hypoxia-responsive genes involved in angiogenesis, inflammation, and metabolism. The HIF/VEGF pathway plays an important role in neovascularization, and the inhibition of HIF activation could be an effective biomolecular target for neovascular diseases. The demand for disease prevention or treatment using functional foods such as superfoods has increased in recent years. Few reports to date have focused on the antineovascular effects of superfoods in the retinal pigment epithelium (RPE). In light of the growing demand for functional foods, we aimed to find novel HIF inhibitors from superfoods worked in RPE cells, which could be an adjuvant for anti-VEGF therapy. Seven superfoods were examined to identify novel HIF inhibitor candidates using luciferase assay screening. We used the human RPE cell line ARPE-19 and fetal human RPE (fhRPE) to investigate the biomolecular actions of novel HIF inhibitors using quantitative PCR and western blotting. Under CoCl2-induced pseudohypoxic condition and 1% oxygen hypoxic incubation, camu-camu (Myrciaria dubia) showed HIF inhibitory effects determined by luciferase assays. Camu-camu downregulated HIF-1α and VEGFA mRNA expressions in a concentration-dependent manner. Camu-camu also inhibited HIF-1α protein expressions, and its inhibitory effect was greater than that of vitamin C, which is present at high levels in camu-camu. The camu-camu extract suppressed the activation of HIF and VEGF in RPE cells. This could assist anti-VEGF therapy in patients with abnormal ocular neovascularization.

The effect of natural products combination on MCF-7 cells exceeds tamoxifen therapeutic dose effects in vitro

Cancer remains to be one of the most severe sicknesses globally. Cases have kept rising over the years. Breast cancer (BC), which is among the leading types of cancers and predominantly affects women, is the second leading cause of cancer mortality. Researchers have developed interventions over the years; however, the BC survival rate has not improved since the 1980s. This has created the need for novel drug interventions that would manage and treat BC more effectively. This study focused on using a combination of natural product extracts such as phytoestrogen (Ziziphus jujube) and Tannin nanoparticles (NP99) together, which we have referred to as (Z.NP99) and tamoxifen (Tam) as one of the leading BC drugs since the 70s, in treating BC. The effectiveness of Tam if used alone in the treatment and if combined with Z.NP99 was evaluated using MCF-7 cells in vitro. The findings showed that the combination treatment of Z.NP99 affected the proliferation and viability of MCF-7 cells more than Tam at a 10 μg/mL dose. Moreover, Z.NP99 with Tam stimulated the maximum reduction of MCF-7 proliferation and viability in a time-dependent manner. Furthermore, Tam and Z.NP99 augmented the DNA fragmentation percentage combined with the upregulation of the apoptotic genes. Additionally, the results showed that the apoptotic impact of Z.NP99 and Tam on MCF-7 cells may be intermediated by down-regulating some genes such as Claudin-1 followed by down-regulating mRNA expression of MMP-9, VEGF, and BCL-2 genes of treated cells. Combining Tam with Z.NP99 considerably enhanced the effectiveness of conventional therapy. As a result, this study suggested that the Z.NP99 was ideal for developing effective natural treatments that would improve BC outcomes.

Sidaxue, a traditional Guizhou Miao herbal medicine formula, reduces necroptosis and synovial vascular proliferation in rats with collagen-induced arthritis by inhibiting the RIPK1/RIPK3/MLKL pathway

To explore the inhibitory effect of Sidaxue (SX), a traditional Guizhou Miao herbal medicine formula, on necrotic apoptosis and synovial angiogenesis in rats with collagen-induced arthritis (CIA) and the role of the RIPK1/RIPK3/MLKL pathway in mediating this effect. Forty-two SD rats were randomized into 6 groups (n=7), including a normal control group, a CIA model group, 3 SX treatment groups at low (10 g/kg), moderate (20 g/kg) and high (40 g/kg) doses, and a GTW treatment group. CIA rat models were established by subcutaneous injections of bovine type II collagen, and the treatments were administered daily by gavage for 21 days. The rats were observed for swelling of the hind limb joints, which was rated using the arthritis index (AI) score on a weekly basis. Serum levels of TNF-α, IL-1β and IL-17 in the rats were detected using ELISA, and the pathological changes in the synovium were observed with HE staining. Real-time PCR was performed to detect the mRNA expression levels of VEGF, MMP-9, Ang-1, RIPK1, RIPK3, and caspase-8 in the synovial tissues, and the protein expressions of VEGF, MMP9, Ang-1, Stat-3, RIPK1, RIPK3, MLKLl, p-MLKL and caspase-8 were detected using Western blotting. Compared with those in CIA model group, the rats receiving treatment with GTW and SX showed milder swelling of the hind limb joints with significantly lower AI scores (P < 0.05). In CIA model group, a large number of inflammatory cells were observed in the synovium with obvious damages of the tissue structure. In the drug treatment groups, inflammatory cell infiltration, synovial angiogenesis and synovial hyperplasia were alleviated, and the therapeutic effects were obviously enhanced as SX dose increased. Compared with those in the model group, the rats treated with GTW and high-dose SX showed significantly decreased serum levels of IL-1β, IL-17 and TNF-α (P < 0.05), lower mRNA and protein expressions of RIPK1, RIPK3, VEGF, Ang-1, and MMP9 (P < 0.05), higher expressions of caspase-8 (P < 0.01), and obviously lowered expression of Stat-3 protein and phosphorylation level of MLKL (P < 0.05). SX can improve synovial angiogenesis in CIA rats possibly by inhibiting the activation of RIP1/RIP3/MLKL signaling pathway and down-regulating the expression of the vascular growth factors VEGF, Ang-1, MMP9, and Stat-3.

Ternary regulation mechanism of Rhizoma drynariae total flavonoids on induced membrane formation and bone remodeling in Masquelet technique.

Rhizoma drynariae total flavonoids (RDTF) are used to treat fractures. CD31hiEmcnhi vessels induced by PDGF-BB secreted by osteoclast precursors, together with osteoblasts and osteoclasts, constitute the ternary regulatory mechanism of bone tissue reconstruction. This study aimed to determine whether RDTF can promote bone tissue remodeling and induce membrane growth in the rat Masquelet model and to explore its molecular mechanism based on the ternary regulation theory. Thirty-six SD rats were randomized to three groups: blank, induced membrane, and RDTF treatment (n = 12/group). The gross morphological characteristics of the new bone tissue were observed after 6 weeks. Sixty SD rats were also randomized to five groups: blank, induction membrane, low-dose RDTF, medium-dose RDTF, and high-dose RDTF (n = 12/group). After 4 weeks, immunohistochemistry and western blot were used to detect the expression of membrane tissue-related proteins. The mRNA expression of key factors of ternary regulation was analyzed by qRT-PCR. RDTF positively affected angiogenesis and bone tissue reconstruction in the bone defect area. RDTF could upregulate the expression of key factors (PDGF-BB, CD31, and endomucin), VEGF, and HMGB1 mRNA and proteins in the ternary regulation pathway. Although the expected CD31hiEmcnhi vessels in the induction membrane were not observed, this study confirmed that RDTF could promote the secretion of angiogenic factors in the induced membrane. The specific mechanisms still need to be further studied.