VEGF Protein Expression Levels Research Articles

Batroxase promotes the effect of NK cell adoptive therapy on lung cancer by enhancing immune cell infiltration.

Batroxobin, isolated from Bothrops moojeni, is a defibrinogenating agent used as a thrombin-like serine protease against fibrinogen for improving microcirculation. Here, we investigated whether, and if so, how batroxobin acts in concert with NK cells in terms of anti-tumor effects. CD3+/CD56+ NK cells were isolated and cultured from C57BL/6 mouse spleen. NK cells' viability was tested via Lactate dehydrogenase (LDH) assay. Lewis lung cancer cell (1*107 cell/ml) was used to build animal models. All animals were divided into five groups and treated with Batroxobin and NK cells respectively. HE staining was used to detect the pathological morphology of tumor tissue. The contents of fibrinogen and TNF-α in serum were determined by ELISA. The protein expression levels of MMP2, MMP9, VEGF and CD44 in tumor tissues were detected by Western Blot or immunohistochemistry. Compared with Control group, Tumor growth was not significantly affected in the group treated with Batroxobin or NK cells alone, However, tumor growth was significantly inhibited in the NK cell combined with the Batroxobin group. Serum levels of Fbg and TNF-αin mice treated with Batroxobin combined with NK cells dropped significantly, bringing them closer to normal levels. WB results showed that the expression levels of MMP2/9, VEGF and CD44 in Batroxobin combined with NK cell group also significantly decreased. Batroxobin combined with adoptive immunotherapy with NK cells significantly inhibited the growth of Lewis lung cancer in mice.

Effect of RNA interference with HIF-1α on the growth of pulmonary artery endothelial cells in broiler chickens

Pulmonary artery remodeling is a characteristic feature of broiler ascites syndrome (BAS). Pulmonary artery endothelial cells (PAECs) regulated by HIF-1α play a critical role in pulmonary artery remodeling, but the underlying mechanisms of HIF-1α in BAS remain unclear. In this experiment, primary PAECs were cultured in vitro and were identified by coagulation factor VIII. After hypoxia and RNA interference, the mRNA and protein expression levels of HIF-1α and VEGF were determined by qPCR and Western blotting. The transcriptome profiles of PAECs were obtained by RNA sequencing. Our results showed that the positive rate of PAECs was more than 90%, hypoxia-induced promoted the proliferation and apoptosis of PAECs, and RNA interference significantly downregulated the expression of HIF-1α, inhibited the proliferation of PAECs, and promoted the apoptosis of PAECs. In addition, transcriptome sequencing analysis indicated that HIF-1α may regulate broiler ascites syndrome by mediating COL4A, vitronectin, vWF, ITGα8, and MKP-5 in the ECM, CAMs and MAPK pathways in PAECs. These studies lay the foundation for further exploration of the mechanisms of pulmonary artery remodeling, and HIF-1α may be a potentially effective gene for the prevention and treatment of BAS.

Dihydroartemisinin inhibits angiogenesis in breast cancer via regulating VEGF and MMP-2/-9.

Dihydroartemisinin (DHA) is an artemisinin derivative known for its antimalarial properties. It has also shown potential as an anti-tumor and anti-angiogenic agent. However, its specific role in inhibiting angiogenesis in breast cancer is not well understood. We aimed to investigate the anti-angiogenesis effect of DHA on breast cancer and explore its potential as a therapeutic drug. Our objectives were to assess the impact of DHA on neovascularization induced by MDA-MB-231 cells, evaluate its effects on vessel sprout and tube-formation in vascular endothelial cells, and analyze the expression of key angiogenesis-related proteins. Using a chicken chorioallantoic membrane (CAM) model, we cultured MDA-MB-231 cells and treated them with DHA. We assessed neovascularization and cultured vascular endothelial cells with DHA-treated cell media to evaluate vessel sprout and tube-formation. Protein expression levels of VEGF, MMP-2, and MMP-9 were analyzed using Western blotting. DHA significantly attenuated neovascularization induced by MDA-MB-231 cells. It also suppressed vessel sprout and tube-formation of HUVEC cells when exposed to DHA-treated cell media. Furthermore, DHA downregulated the expression of VEGF, MMP-2, and MMP-9 proteins. Mechanistically, DHA inhibited the phosphorylation of PI3K, AKT, ERK, and NF-κB proteins in tumor cells. Our study provides evidence of the inhibitory effect of DHA on breast cancer angiogenesis. These findings support the potential of DHA as an anti-breast cancer drug and warrant further investigation for its therapeutic applications.

Alcohol extracts from Anemoneflaccida Fr. Schmidt treat rheumatoid arthritis via inhibition of synovial hyperplasia and angiogenesis.

Anemoneflaccida Fr. Schmidt, a Traditional Chinese Medicine, has been used in the treatment of rheumatoid arthritis (RA) for numerous years. However, the specific mechanisms remain to be elucidated. Thus, the present study aimed to investigate the main chemical constituents and potential mechanisms of Anemoneflaccida Fr. Schmidt. The ethanol extract obtained from Anemoneflaccida Fr. Schmidt (EAF) was analyzed using mass spectrometry to determine the main components and the therapeutic effects of EAF on RA were verified using a collagen‑induced arthritis (CIA) rat model. Results of the present study demonstrated that synovial hyperplasia and pannus of the model rats were significantly improved following EAF treatment. Moreover, the protein expression levels of VEGF and CD31‑labeled neovascularization were significantly reduced in the synovium of CIA rats following treatment with EAF, compared with those of the untreated model group. Subsequently, invitro experiments were carried out to verify the impact of EAF on synovial proliferation and angiogenesis. Results of the western blot analysis revealed that EAF inhibited the PI3K signaling pathway in endothelial cells, which is associated with anti‑angiogenesis. In conclusion, results of the present study demonstrated the therapeutic effects of Anemoneflaccida Fr. Schmidt on RA and preliminarily revealed the mechanisms of this drug in the treatment of RA.

VEGF regulates the blood‑brain barrier through MMP‑9 in a rat model of traumatic brain injury.

Blood-brain barrier (BBB) damage is closely related to morbidity and mortality in patients with traumatic brain injury (TBI). Inhibition of VEGF effectively protects BBB integrity in clinical ischemic stroke. Protecting BBB integrity, reducing brain edema and alleviating post-TBI secondary brain injury are key to a favorable patient prognosis. MMP-9 affects BBB integrity by destroying the tight junction of vascular endothelial cells and inhibiting the transport and enzymatic systems. The present study aimed to examine the possible interplay between VEGF and MMP-9 in TBI. A TBI model was established in 87 male Sprague-Dawley rats. Reverse transcription-quantitative PCR, western blotting, wet-dry brain edema assessment, TUNEL and Fluoro-Jade C staining were performed to analyze the brain tissue samples of the rats. The results showed that compared with in the Sham group rats, the mRNA and protein expression levels of VEGF and MMP-9 were increased at 24 h post-TBI. After bevacizumab treatment, BBB permeability and nerve cell apoptosis were markedly reduced. In conclusion, the present study revealed a potential role for TBI-associated VEGF and MMP-9 upregulation in BBB disruption and nerve damage post-TBI.

HIF-2α regulates proliferation, invasion, and metastasis of hepatocellular carcinoma cells via VEGF/Notch1 signaling axis after insufficient radiofrequency ablation.

Background and AimsAlthough insufficient radiofrequency ablation (RFA) promotes the recurrence and metastasis of liver cancer, the underlying mechanism remains unclear. This study aimed to investigate the role and mechanism of HIF-2α in hepatocellular carcinoma cells (HCCs) after Insufficient RFA.MethodsWe established a model of insufficient RFA in MHCC97H hepatoma cells and screened for stable sublines. We inhibited HIF-2α expression in the Insufficient RFA group using PT2385 and assessed the resulting changes in proliferation and biological function of HCCs. Cell viability and proliferation were detected by the MTT method, and scratch and Transwell chamber invasion tests detected migration and invasion abilities of HCCs. The mRNA and protein expression levels of VEGF, HIF-2α, and Notch1 were detected using qPCR, immunofluorescence, and western blotting.ResultsCompared with normal HCCs without RFA treatment, insufficient RFA enhanced the proliferation and invasion abilities of hepatocellular carcinoma subline MHCC97H (P < 0.001), as well as their migration ability (P = 0.046). The HIF-2α-specific inhibitor PT2385 downregulated the migration (P = 0.009) and invasion (P < 0.001) of MHCC97H cells but did not affect cell proliferation (P > 0.05). Insufficient ablation increased the mRNA and protein expression of VEGF, HIF-2α, and Notch1 in HCCs, whereas inhibition of HIF-2α reversed these changes.ConclusionsInsufficient RFA increases the proliferation, migration, and invasion of HCCs via the HIF-2α/VEGF/Notch1 signaling axis; HIF-2α is a potential target for novel treatments of HCC after insufficient RFA.

Allyl Isothiocyanate (AITC) Induces Apoptotic Cell Death In Vitro and Exhibits Anti-Tumor Activity in a Human Glioblastoma GBM8401/luc2 Model.

Some clinically used anti-cancer drugs are obtained from natural products. Allyl isothiocyanate (AITC), a plant-derived compound abundant in cruciferous vegetables, has been shown to possess an anti-cancer ability in human cancer cell lines in vitro, including human brain glioma cells. However, the anti-cancer effects of AITC in human glioblastoma (GBM) cells in vivo have not yet been examined. In the present study, we used GBM8401/luc2 human glioblastoma cells and a GBM8401/luc2-cell-bearing animal model to identify the treatment efficacy of AITC. Here, we confirm that AITC reduced total cell viability and induced cell apoptosis in GBM8401/luc2 cells in vitro. Furthermore, Western blotting also showed that AITC induced apoptotic cell death through decreased the anti-apoptotic protein BCL-2, MCL-1 expression, increased the pro-apoptotic protein BAX expression, and promoted the activities of caspase-3, -8, and -9. Therefore, we further investigated the anti-tumor effects of AITC on human GBM8401/luc2 cell xenograft mice. The human glioblastoma GBM8401/luc2 cancer cells were subcutaneously injected into the right flank of BALB/c nude mice to generate glioblastoma xenograft mice. The animals were randomly divided into three groups: group I was treated without AITC (control); group II with 0.1 mg/day of AITC; and group III with 0.2 mg/day of AITC every 3 days for 27 days. Bodyweight, and tumor volume (size) were recorded every 3 days. Tumors exhibiting Luc2 intensity were measured, and we quantified intensity using Living Image software on days 0, 12, and 24. After treatment, tumor weight from each mouse was recorded. Tumor tissues were examined for histopathological changes using H&E staining, and we analyzed the protein levels via immunohistochemical analysis. Our results indicate that AITC significantly inhibited tumor growth at both doses of AITC due to the reduction in tumor size and weight. H&E histopathology analysis of heart, liver, spleen, and kidney samples revealed that AITC did not significantly induce toxicity. Body weight did not show significant changes in any experiment group. AITC significantly downregulated the protein expression levels of MCL-1, XIAP, MMP-9, and VEGF; however, it increased apoptosis-associated proteins, such as cleaved caspase-3, -8, and -9, in the tumor tissues compared with the control group. Based on these observations, AITC exhibits potent anti-cancer activity in the human glioblastoma cell xenograft model via inhibiting tumor cell proliferation and the induction of cell apoptosis. AITC may be a potential anti-GBM cancer drug that could be used in the future.

Kisspeptin treatment improves fetal-placental development and blocks placental oxidative damage caused by maternal hypothyroidism in an experimental rat model.

Maternal hypothyroidism is associated with fetal growth restriction, placental dysfunction, and reduced kisspeptin/Kiss1R at the maternal-fetal interface. Kisspeptin affects trophoblastic migration and has antioxidant and immunomodulatory activities. This study aimed to evaluate the therapeutic potential of kisspeptin in the fetal-placental dysfunction of hypothyroid Wistar rats. Hypothyroidism was induced by daily administration of propylthiouracil. Kisspeptin-10 (Kp-10) treatment was performed every other day or daily beginning on day 8 of gestation. Feto-placental development, placental histomorphometry, and expression levels of growth factors (VEGF, PLGF, IGF1, IGF2, and GLUT1), hormonal (Dio2) and inflammatory mediators (TNFα, IL10, and IL6), markers of hypoxia (HIF1α) and oxidative damage (8-OHdG), antioxidant enzymes (SOD1, Cat, and GPx1), and endoplasmic reticulum stress mediators (ATF4, GRP78, and CHOP) were evaluated on day 18 of gestation. Daily treatment with Kp-10 increased free T3 and T4 levels and improved fetal weight. Both treatments reestablished the glycogen cell population in the junctional zone. Daily treatment with Kp-10 increased the gene expression levels of Plgf, Igf1, and Glut1 in the placenta of hypothyroid animals, in addition to blocking the increase in 8-OHdG and increasing protein and/or mRNA expression levels of SOD1, Cat, and GPx1. Daily treatment with Kp-10 did not alter the higher protein expression levels of VEGF, HIF1α, IL10, GRP78, and CHOP caused by hypothyroidism in the junctional zone compared to control, nor the lower expression of Dio2 caused by hypothyroidism. However, in the labyrinth zone, this treatment restored the expression of VEGF and IL10 and reduced the GRP78 and CHOP immunostaining. These findings demonstrate that daily treatment with Kp-10 improves fetal development and placental morphology in hypothyroid rats, blocks placental oxidative damage, and increases the expression of growth factors and antioxidant enzymes in the placenta.

Epigenetic restoration and activation of ERβ: an inspiring approach for treatment of triple-negative breast cancer

BackgroundTriple-negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer. TNBC lacks targeted therapy receptors, rendering endocrine and HER2-targeted therapies ineffective. TNBC is typically treated with cytotoxic chemotherapy followed by surgery. Targeting epigenetic modifications could potentially be a new effective TNBC target therapy. The aim of this study is to examine the effects of epigenetic drugs, decitabine as DNA methyltransferase inhibitor (DNMTI) and vorinostat as histone deacetylase inhibitor (HDACI), and the ERβ agonist DPN on ERα and ERβ re-expressions in the MDA-MB-231 cells as a model of TNBC.MethodsUsing MTT assay, the IC50 of decitabine, vorinostat, and DPN on MDA-MB-231 cells were determined. The effects of all drugs alone or in combinations on MDA-MB-231 cells were evaluated. qRT-PCR was used to determine ERα & ERβ gene expression. Caspase-3 activity and the protein expression levels of VEGF, Cyclin D1, and IGF-1 were assessed.ResultsBoth ERα and ERβ mRNA were re-expressed in different high levels in all treated groups, especially in the triple therapy group compared with control. Significantly, the triple drugs therapy showed the lowest levels of VEGF, Cyclin D1, and IGF-1 and the highest level of Caspase-3 activity, indicating a possible antitumor effect of ERβ activation through decreasing proliferation and angiogenesis and increasing apoptosis in MDA-MB-231 cells.ConclusionsThe antiproliferative effect of ERβ could be retained when co-expressed with Erα using a powerful epigenetic combination of Decitabine and vorinostat with DPN.

Mechanism of ischemic brain injury repair by endothelial progenitor cell‑derived exosomes.

Ischemic stroke is a refractory disease that seriously endangers human health and life. The main treatment aim of stroke is to alleviate brain injury. The present study aimed to investigate the effects and mechanisms of endothelial progenitor cell (EPC)‑derived exosomes in repairing ischemic brain injury. Sprague‑Dawley rat models of cerebral ischemia‑reperfusion (IR) injury were established by middle cerebral artery occlusion. The IR model rats were then treated with PBS, EPC or exosomes; untreated and Sham rats were used as control. EPCs were obtained from tibias and femurs, and exosomes were isolated from the EPCs and characterized. To measure brain injury, 2,3,5‑triphenyltetrazolium chloride staining was used to measure the infarct area, neurological deficit was scored, hematoxylin and eosin staining was used to examine pathological changes and TUNEL staining was used to quantify apoptosis. Immunofluorescence staining and reverse transcription‑quantitative PCR were used to determine CD31 and VEGF protein and mRNA expressions, respectively, and western blot analysis was used out to measure the protein expression levels of Wnt3a, GSK‑3β and phosphorylated (p)‑GSK‑3β. Compared with rats in the Control and Sham groups, in IR model rats the nerve fibers were slightly necrotic and swollen and the number of nerve cells was reduced. Following EPC treatment, the brain tissue exhibited mild liquefaction and degeneration in the small focus area with mild edema in the stroma. The numbers of nerve cells decreased, and the distribution of nerve cells was not very uniform; proliferation of glial cells was observed. Following treatment with exosomes, the distribution of nerve cells was more uniform with less degeneration and necrosis; the proliferation of glial cells was remarkable. Compared with the Control group, the infarct size, neurological defect score, percentage of apoptotic cells, expression of CD31, VEGF, Wnt3a, and p‑GSK‑3β were significantly higher in the IR model (P<0.05). After EPC and exosome treatments, the infarct size, neurological defect score, percentage of apoptotic cells, expression of Wnt3a, and p‑GSK‑3β were significantly reduced (P<0.05), whereas the mRNA and protein expression levels of CD31 and VEGF were significantly increased (P<0.05). Results from the present study demonstrated that EPC‑derived exosomes may alleviate ischemic injury by inhibiting apoptosis and promoting angiogenesis. These findings suggested that exosomes may have a protective role for nerve cells and may be a potentially effective option for treating stroke. However, human clinical studies are needed to validate these findings from animals.

Protective effect of royal jelly on fluoride-induced nephrotoxicity in rats via the some protein biomarkers signalling pathways: a new approach for kidney damage

Introduction Protective effect of royal jelly (RJ) on fluoride-induced nephrotoxicity was investigated in this study. Methods 42 healthy male Wistar rats (n = 42, 8 weeks of age) were divided equally into 6 groups with 7 rats in each; (1) Group-1: Controls fed with standard diet; (2) Group-2: RJ [100 mg/kg] bw (body weight), by oral gavage; (3) Group-3: Fluoride [50 mg/kg] bw, in drinking water; (4) Group-4: Fluoride [100 mg/kg] bw, in drinking water; (5) Group-5: RJ [100 mg/kg] bw, by oral gavage + Fluoride [50 mg/kg] bw, in drinking water; (6) Group-6: RJ [100 mg/kg] bw, by oral gavage + Fluoride [100 mg/kg] bw, in drinking water. After 8 weeks, all rats were decapitated and their kidney tissues were removed for further analysis. The protein expression levels of caspase-3, caspase-6, caspase-9, Bcl-2, Bax, VEGF, GSK-3, BDNF, COX-2 and TNF-α proteins in kidney tissue were analysed by western blotting technique Results RJ increased Bcl-2, COX-2, GSK-3, TNF-α and VEGF protein levels and a decreased caspase-3, caspase −6, caspase-9, Bax and BDNF protein levels in fluoride-treated rats. Conclusion RJ application may have a promising therapeutical potential in the treatment of many diseases in the future by reducing kidney damage.

Effect of low energy shock wave on testicular microenvironment homeostasis in rats

ObjectiveTo further investigate whether two sets of low-energy extracorporeal shock waves (LESWs) impulse parameters, i.e., 0.02 mJ/mm2 for 500 impulses and 0.04 mJ/mm2 for 500 impulses, which have been shown to directly affect the testes, can promote testicular spermatogenesis or positively regulate homeostasis of the testicular microenvironment. Methods(1) Twenty-four experimental rats were randomly divided into a 0.02 mJ/mm2 500 impulses group (L1 group), a 0.04 mJ/mm2 500 impulses group (M1 group), a sham intervention group (S group) and a blank control group (N group). The experiment period was 8 weeks. (2) Apoptosis of the spermatogenic cells in the left testicle was detected by the TUNEL method, VEGF and eNOs protein expression was detected by immunohistochemistry, and histomorphological changes were observed in PAS-stained sections. Moreover, the morphologies of the spermatogenic tubules and testicular stroma were quantitatively analyzed by stereological analysis. The right testicle was used for Western blot detection of the protein expression levels of Bax, Cytochrome C, Caspase-3, Bcl-2, VEGF and eNOs. ResultsCompared with the other three groups, the rate of M1 testicular germ cell apoptosis induced by shock treatment was higher, the expression levels of proapoptotic proteins increased significantly while that of the antiapoptotic protein was lower, and the suppression of cell proliferation correlated with the protein expression levels. Additionally, with respect to the absolute volume of the seminiferous tubules, the absolute interstitial testicular volume notably increased, producing a series of biological effects working against testicular sperm production and function. However, there was no significant difference between the L1 group and the N and S groups. ConclusionsLESWs treatment with impulse parameters of 0.02 mJ/mm2 for 500 impulses showed a better protective effect on testicular spermatic function in rats and has a positive regulatory biological effect.

Irbesartan protects against type 2 diabetic nephropathy by regulating miR-93/VEGF and its downstream effectors

Purpose: To investigate the protective effect of irbesartan against type 2 diabetic nephropathy (T2DN), and the mechanism involved.Methods: Wistar rats (n = 48) were used in this study. Diabetes mellitus (DM) was established in the rats via injection of streptozotocin (STZ). Thereafter, the DM rats were randomly divided into diabetic nephropathy (DN) group and irbesartan group, with 16 rats in each group. Rats in the control group (n = 16) received normal saline in place of irbesartan.Results: Total cholesterol (TC), triglycerides (TGs), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C), as well as protein expression levels of VEGF, FN, collagen IV in DN group were significantly higher in irbesartan group than the corresponding levels in normal control group, while miR-93 protein expression level was significantly lower than that in normal control group (p &lt; 0.05). However, the expression level of mir-93 was significantly higher in irbesartan group than in DN group, while levels of TC, TGs, HDL-C, LDL-C and VEGF, as well as protein levels of FN and collagen IV protein were significantly lower than those in DN group (p &lt; 0.05). There was no obvious change in the renal tissues of the normal control group. In contrast, in the DN group, glomerularcapillary loop hypertrophy, narrow glomerular cavity, thick basement membrane, mesangial matrix, and vacuolar degeneration in renal tubular epithelial cells, were evident. Compared with DN group, the pathological changes in the irbesartan group were significantly mitigated (p &lt; 0.05).Conclusion: Irbesartan protects DM rats against type 2 diabetic nephropathy by regulating mir-93/VEGF and its downstream effector molecules. This provides some ideas for the development of new drugs for the prevention of type 2 diabetic nephropathy.

Exosomal miRNA-200b-3p regulated autogenous arteriovenous fistula thrombosis in maintenance hemodialysis patients.

Autogenous arteriovenous fistula (AVF) is the best vascular hemodialysis access for terminal chronic renal failure patients but is prone to thrombosis. Pathogenic mechanisms of AVF thrombus are thus largely explored. As exosomes carry genetic content from cell of origin. We hypothesized that miRNAs in serum exosomes are promising regulators of AVF thrombosis. Serum exosomes were isolated from maintenance hemodialysis (MHD) patient, miRNAs profile of the exosomes was obtained by high throughput sequencing, six miRNAs (miR-144-5p, miR-18a-5p, miR-200a-3p, miR-200b-3p, miR-141-3p, and miR-429) were determined as candidates examined by RT-PCR, cells transfected with miR-200b-3p mimics demonstrated significantly increased mRNA levels of VEGF and Ang-II, the relationship between miR-200b-3p and VEGF or Ang-II was performed by adual luciferase reporter assay. There are 43 miRNA down-regulation and 15 miRNA up-regulation between MHD group and MHD+Thrombus group, the expression levels of miR-200b-3p and miR-429 in MHD with thrombus were significantly increased (p < 0.001, p < 0.05). Inhibited miR-200b-3p expression level can increase VEGF mRNA and protein expression levels and decrease Ang-II mRNA and protein expression levels. Furthermore, we also identified that miR-200b-3p targets VEGF and Ang-II. Our study indicates that serum exosome-derived miR-200b-3p regulate VEGF and Ang-II to increase intimal hyperplasia to induce AVF thrombosis. Besides miR-200b-3p, miR-200 family may also play a regulatory role in AVF thrombosis.

Effects of miR-363 on the Biological Activities of Eutopic Endometrial Stromal Cells in Endometriosis.

EMs is a kind of benign disease with certain malignant behaviors. The adhesion, invasive growth, and angiogenesis of ectopic endometrial cells are the pathological basis of EMs occurrence, but its etiology and pathogenesis have not been completely illustrated yet. In our research, we aim to investigate the role of miR-363 in the pathogenesis of endometriosis. Real-time quantitative PCR was used to detect the expression of miR-363 before and after ESC/NSC transfection. CCK-8, flow cytometry, and transwell assay were used to detect the effect of the miR-363 expression on cell proliferation, apoptosis, and invasion. The effects of the miR-363 expression on the contents of Fas/APO-1 and ICAM-1 in cell culture supernatant were detected by ELISA. qRT-PCR and WB assay were used to detect the effects of the miR-363 expression on the mRNA and protein expression levels of ICAM-1, MMP-7, and VEGF in ESC. The increased expression of miR-363 could inhibit the proliferation and invasion of ESC, promote apoptosis, and inhibit the secretion of FAS/APO-1 and ICAM-1. The knockdown expression of miR-363 promoted proliferation and invasion of NSC, inhibited apoptosis, and promoted secretion of FAS/APO-1 and ICAM-1. VCAM-1, VEGF, and MMP-7 were detected in ESCs before transfection. The protein expression level was higher than that of NSCs. Compared with pretransfection, the protein levels of VCAM-1, VEGF, and MMP-7 in the M-363 group were significantly downregulated. The downregulated expression of miR-363 was associated with a stronger cell proliferation ability, a lower cell apoptosis rate, and a stronger ESC. Migration is associated with invasiveness, proliferation, angiogenesis, and immune escape. The low expression of miR-363 promotes endogenesis through posttranscriptional regulation of target genes VCAM-1, MMP-7, and VEGF. The differential expression of miR-363 between ESC and NSC may be an important factor in the many biological differences between ESC and NSC.

Ascorbic acid 2-glucoside preconditioning enhances the ability of bone marrow mesenchymal stem cells in promoting wound healing

BackgroundNowadays, wound is associated with a complicated repairing process and still represents a significant biomedical burden worldwide. Bone marrow mesenchymal stem cells (BMSCs) possess multidirectional differentiation potential and secretory function, emerging as potential cellular candidates in treating wounds. Ascorbic acid 2-glucoside (AA2G) is a well-known antioxidant and its function in BMSC-promoting wound healing is worth exploring.MethodsThe in vitro cell proliferation, migration, and angiogenesis of BMSCs and AA2G-treated BMSCs were detected by flow cytometry, EDU staining, scratch assay, transwell assay, and immunofluorescence (IF). Besides, the collagen formation effect of AA2G-treated BMSCs conditioned medium (CM) on NIH-3T3 cells was evaluated by hydroxyproline, qRT-PCR and IF staining detection. Next, in the wound healing mouse model, the histological evaluation of wound tissue in PBS, BMSCs, and AA2G-treated BMSCs group were further investigated. Lastly, western blot and ELISA were used to detect the expression levels of 5-hmc, TET2 and VEGF protein, and PI3K/AKT pathway activation in BMSCs treated with or without AA2G.ResultsThe in vitro results indicated that AA2G-treated BMSCs exhibited stronger proliferation and improved the angiogenesis ability of vascular endothelial cells. In addition, the AA2G-treated BMSCs CM enhanced migration and collagen formation of NIH-3T3 cells. In vivo, the AA2G-treated BMSCs group had a faster wound healing rate and a higher degree of vascularization in the new wound, compared with the PBS and BMSCs group. Moreover, AA2G preconditioning might enhance the demethylation process of BMSCs by regulating TET2 and up-regulating VEGF expression by activating the PI3K/AKT pathway.ConclusionsAA2G-treated BMSCs promoted wound healing by promoting angiogenesis and collagen deposition, thereby providing a feasible strategy to reinforce the biofunctionability of BMSCs in treating wounds.

The effect of miR-138 on the proliferation and apoptosis of breast cancer cells through the NF-κB/VEGF signaling pathway.

The analyze the effect of miR-138 on the proliferation and apoptosis of breast cancer cells through the NF-κB/VEGF signaling pathway is the Objective of this experiment. For this aim, the endometrial stem breast cancer cell line MCF-7 was cultured in vitro, and the overexpression mimic miR-138 mimics and the inhibitor anti-miR-138 were transfected into the endometrial stem breast cancer cell line MCF-7, which was set to overexpress miR-138 group and interfere with miR-138, and set up negative control of overexpression and negative control of inhibitor. Observe the cell proliferation and apoptosis ability of each group, and the changes in tumor necrosis factor-α (TNF-α), interleukin 1β, 6, 18 (IL-1β, IL-6, IL-18) levels, and compare the Bax of each group, NF-κB, VEGF protein expression level. Results showed that the proliferation ability of the miR-138 overexpression group was significantly lower than that of the miR-138 overexpression control group (P<0.05); the proliferation ability of the miR-138 interference group was significantly higher than that of the miR-138 interference control group (P<0.05). The apoptosis rate, caspase-3 and caspase-9 expression levels of the miR-138 overexpression group were significantly higher than those of the miR-138 overexpression control group (P<0.05); the apoptosis rate, caspase-3 and caspase-9 expression levels of the miR-138 interference group were significantly lower than those of the miR-138 interference control group (P<0.05). The expression levels of IL-1 β, IL-6, IL-18 and TNF - α in the miR-138 overexpression group were significantly lower than those in the miR-138 overexpression control group (P < 0.05). The protein expression levels of Bax, NF-κB and VEGF in the miR-138 overexpression group were significantly lower than those in the miR-138 overexpression control group (P < 0.05); the protein expression levels of Bax, NF-κB and VEGF in the miR-138 interference group were significantly higher than those in the miR-138 interference control group (P <0.05). The proliferation ability of the miR-138 overexpression group was significantly lower than that of the miR-138 overexpression control group (P < 0.05); the proliferation ability of the miR-138 + NF-κB overexpression group was significantly higher than that of the miR-138 overexpression group (P<0.05). The apoptosis rate of the miR-138 + NF-κB overexpression group was significantly lower than that of the miR-138 overexpression group (P < 0.05). Then MiR-138 can significantly inhibit the proliferation of breast cancer cells, promote apoptosis, and regulate the expression of inflammatory factors in the cells. It is speculated that the related mechanism may be related to the negative regulation of the NF-κB/VEGF signaling pathway.

Pyruvate kinase M2 (PKM2) improve symptoms of post-ischemic stroke depression by activating VEGF to mediate the MAPK/ERK pathway.

PurposeTo evaluate and identify the effects and explore the mechanisms of pyruvate kinase M2 (PKM2) on stroke‐induced post stroke depression (PSD).MethodsRats were separated into six different groups, including sham + saline, Stroke + saline, PSD + saline, PSD + recombinant pyruvate kinase M2 (rPKM2) (112 ng/kg), PSD + rPKM2 (224 ng/kg), and PSD + rPKM2 (224 ng/kg) + bevacizumab. Then, the body weight, sucrose preference rate, immobility time, horizontal movement, and vertical movement were determined to evaluate the effect of PKM2 on improving the depressive behavior of PSD rats. Subsequently, the proliferation of oligodendrocytes in subventricular zone (SVZ) of rats in each group was examined by western blot and immunofluorescent staining. Furthermore, the mRNA and protein expression levels of TNF‐α, IL‐6, and IL‐1β were also detected by qPCR and ELISA to verify the anti‐inflammatory effects of PKM2 on PSD rats. In addition, the protein expression levels of MDA, LDH, and NO were tested to reveal that PKM2 can reduce oxidative stress in PSD rats. The western blot and IHC assays were employed to examine the protein expression levels of VEGF, PKM2, and ERK in PSD rats.ResultsIn this study, the results showed that PKM2 can improve the depressive behavior and proliferation of oligodendrocytes in PSD rats. In addition, PKM2 has anti‐inflammatory and anti‐oxidative stress effects on PSD rats. Meanwhile, PKM2 activated the expression level of VEGF/MAPK/ERK pathway.ConclusionPKM2 improves symptoms of post‐ischemic stroke depression by activating VEGF‐mediated MAPK/ERK pathway.

VEGF mitigates bisphosphonate-induced apoptosis and differentiation inhibition of MC3T3-E1 cells.

The present study aimed to investigate whether VEGF was involved in bisphosphonate (BP)-induced apoptosis and differentiation of osteoblasts. Murine MC3T3-E1 osteoblasts were stimulated with zoledronic acid (ZA) for 7 days. VEGF mRNA and protein expression levels were determined via reverse transcription-quantitative PCR and western blot analysis, respectively. Cell viability was evaluated using Cell Counting Kit-8 assay. In addition, the cell apoptotic rate and the expression levels of apoptosis-related proteins were measured using a TUNEL staining kit and western blot analysis, respectively. To evaluate mineralization, cells were stained with alizarin red, while the secretion levels of alkaline phosphatase (ALP) were measured using the corresponding assay kit. Finally, the expression levels of differentiation-related proteins and proteins of the Nod-like receptor family pyrin domain-containing 3 (NLRP3)/caspase 1/gasdermin D (GSDMD) pyroptosis pathway were measured by western blot analysis. VEGF expression level was notably decreased in ZA-stimulated MC3T3-E1 cells. However, the viability of these cells was enhanced following VEGF addition. Furthermore, VEGF attenuated apoptosis, promoted mineralization and increased ALP activity in ZA-stimulated MC3T3-E1 cells. The ZA-mediated decrease in the protein expression of the osteogenic genes osteopontin, osteocalcin and runt-related transcription factor 2 was restored after MC3T3-E1 cell treatment with 10 ng/ml VEGF. The present study demonstrated that VEGF could attenuate BP-induced apoptosis and differentiation of MC3T3 cells by regulating the NLRP3/caspase 1/GSDMD pathway.

Garlic (Allium sativum)-derived SEVs inhibit cancer cell proliferation and induce caspase mediated apoptosis

As a key component of the cell-to-cell communication, small extracellular vesicles (SEVs) released from various sources are known to be affecting the physiological conditions of the target cells. Although it has been suggested that edible plant-derived nanoparticles contributes to the cross kingdom communication with the mammalian cells, the effect of these particles on cancer cell progression still needs a further exploration. Here, we isolated and then characterized garlic derived SEVs by nanoparticle tracking analysis, electron microscopy and SEV surface antibodies. In order to investigate anti-cancer property of garlic SEVs A498 human kidney carcinoma, A549 human lung carcinoma were used as cell models along with the normal human dermal fibroblast cell lines. Annexin V/pI staining and analysis of apoptotic mRNA and protein expression levels suggested that garlic SEVs induced apoptosis through activation of intrinsic pathway. Furthermore, angiogenic VEGF protein expression levels significantly decreased in response to SEVs treatment in cancer cells. Our results support that garlic derived SEVs could cause apoptotic cell death among cancer cells while normal cells remain unaffected with the treatment. This study revealed for the first time that plant SEVs possess anti-cancer affects by inducing caspase mediated apoptosis and provided a new alternative for cancer treatment.