OBJECTIVE: To investigate the effects of Danmu Extract Syrup (DMS) on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice and explore the mechanism. METHODS: Seventy-two male Balb/C mice were randomly divided into 6 groups according to a random number table (n=12), including control (normal saline), LPS (5 mg/kg), LPS+DMS 2.5 mL/kg, LPS+DMS 5 mL/kg, LPS+DMS 10 mL/kg, and LPS+Dexamethasone (DXM, 5 mg/kg) groups. After pretreatment with DMS and DXM, the ALI mice model was induced by LPS, and the bronchoalveolar lavage fluid (BALF) were collected to determine protein concentration, cell counts and inflammatory cytokines. The lung tissues of mice were stained with hematoxylin-eosin, and the wet/dry weight ratio (W/D) of lung tissue was calculated. The levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1 β in BALF of mice were detected by enzyme linked immunosorbent assay. The expression levels of Claudin-5, vascular endothelial (VE)-cadherin, vascular endothelial growth factor (VEGF), phospho-protein kinase B (p-Akt) and Akt were detected by Western blot analysis. RESULTS: DMS pre-treatment significantly ameliorated lung histopathological changes. Compared with the LPS group, the W/D ratio and protein contents in BALF were obviously reduced after DMS pretreatment (P<0.05 or P<0.01). The number of cells in BALF and myeloperoxidase (MPO) activity decreased significantly after DMS pretreatment (P<0.05 or P<0.01). DMS pre-treatment decreased the levels of TNF-α, IL-6 and IL-1 β (P<0.01). Meanwhile, DMS activated the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathway and reversed the expressions of Claudin-5, VE-cadherin and VEGF (P<0.01). CONCLUSIONS DMS attenuated LPS-induced ALI in mice through repairing endothelial barrier. It might be a potential therapeutic drug for LPS-induced lung injury.
Mice ; Male ; Animals ; Proto-Oncogene Proteins c-akt/metabolism* ; Lipopolysaccharides ; Phosphatidylinositol 3-Kinases/metabolism* ; Interleukin-1beta/metabolism* ; Vascular Endothelial Growth Factor A/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Claudin-5/metabolism* ; Acute Lung Injury/chemically induced* ; Lung/pathology* ; Interleukin-6/metabolism* ; Drugs, Chinese Herbal

Hypoxia-inducible factor (HIF-1α), a core transcription factor responding to changes in cellular oxygen levels, is closely associated with a wide range of physiological and pathological conditions. However, its differential impacts on vascular cell types and molecular programs modulating human vascular homeostasis and regeneration remain largely elusive. Here, we applied CRISPR/Cas9-mediated gene editing of human embryonic stem cells and directed differentiation to generate HIF-1α-deficient human vascular cells including vascular endothelial cells, vascular smooth muscle cells, and mesenchymal stem cells (MSCs), as a platform for discovering cell type-specific hypoxia-induced response mechanisms. Through comparative molecular profiling across cell types under normoxic and hypoxic conditions, we provide insight into the indispensable role of HIF-1α in the promotion of ischemic vascular regeneration. We found human MSCs to be the vascular cell type most susceptible to HIF-1α deficiency, and that transcriptional inactivation of ANKZF1, an effector of HIF-1α, impaired pro-angiogenic processes. Altogether, our findings deepen the understanding of HIF-1α in human angiogenesis and support further explorations of novel therapeutic strategies of vascular regeneration against ischemic damage.
Humans ; Vascular Endothelial Growth Factor A/metabolism* ; Endothelial Cells/metabolism* ; Transcription Factors/metabolism* ; Gene Expression Regulation ; Hypoxia/metabolism* ; Cell Hypoxia/physiology*

OBJECTIVES: This study aims to investigate the effects of tumor-stromal fibroblasts (TSFs) on the proliferation, invasion, and migration of salivary gland pleomorphic adenoma (SPA) cells in vitro. METHODS: Salivary gland pleomorphic adenoma cells (SPACs), TSFs, and peri-tumorous normal fibroblasts (NFs) were obtained by tissue primary culture and identified by immunocytochemical staining. The conditioned medium was obtained from TSF and NF in logarithmic phase. SPACs were cultured by conditioned medium and treated by TSF (group TSF-SPAC) and NF (group NF-SPAC). SPACs were used as the control group. The proliferation, invasion, and migration of the three groups of cells were detected by MTT, transwell, and scratch assays, respectively. The expression of vascular endothelial growth factor (VEGF) in the three groups was tested by enzyme linked immunosorbent assay (ELISA). RESULTS: Immunocytochemical staining showed positive vimentin expression in NF and TSF. Results also indicated the weak positive expression of α-smooth muscle actin (SMA) and fibroblast activation protein (FAP) in TSFs and the negative expression of α-SMA and FAP in NFs. MTT assay showed that cell proliferation in the TSF-SPAC group was significantly different from that in the NF-SPAC and SPAC groups (P<0.05). Cell proliferation was not different between the NF-SPAC and SPAC groups (P>0.05). Transwell and scratch assays showed no difference in cell invasion and migration among the groups (P>0.05). ELISA showed that no significant difference in VEGF expression among the three groups (P>0.05). CONCLUSIONS TSFs may be involved in SPA biological behavior by promoting the proliferation of SPACs but has no effect on the invasion and migration of SPACs in vitro. Hence, TSF may be a new therapeutic target in SPA treatment.
Humans ; Adenoma, Pleomorphic/metabolism* ; Vascular Endothelial Growth Factor A ; Culture Media, Conditioned/metabolism* ; Fibroblasts/metabolism* ; Salivary Glands/metabolism*

Diabetic ulcer(DU) is one of the common complications of diabetes often occurring in the peripheral blood vessels of lower limbs or feet with a certain degree of damage. It has high morbidity and mortality, a long treatment cycle, and high cost. DU is often clinically manifested as skin ulcers or infections in the lower limbs or feet. In severe cases, it can ulcerate to the surface of tendons, bones or joint capsules, and even bone marrow. Without timely and correct treatment, most of the patients will have ulceration and blackening of the extremities. These patients will not be able to preserve the affected limbs through conservative treatment, and amputation must be performed. The etiology and pathogenesis of DU patients with the above condition are complex, which involves blood circulation interruption of DU wound, poor nutrition supply, and failure in discharge of metabolic waste. Relevant studies have also confirmed that promoting DU wound angiogenesis and restoring blood supply can effectively delay the occurrence and development of wound ulcers and provide nutritional support for wound healing, which is of great significance in the treatment of DU. There are many factors related to angiogenesis, including pro-angiogenic factors and anti-angiogenic factors. The dynamic balance between them plays a key role in angiogenesis. Meanwhile, previous studies have also confirmed that traditional Chinese medicine can enhance pro-angiogenic factors and down-regulate anti-angiogenic factors to promote angiogenesis. In addition, many experts and scholars have proposed that traditional Chinese medicine regulation of DU wound angiogenesis in the treatment of DU has broad prospects. Therefore, by consulting a large number of studies available, this paper expounded on the role of angiogenesis in DU wound and summarized the research advance in traditional Chinese medicine intervention in promoting the expression of angiogenic factors [vascular endothelial growth factor(VEGF), fibroblast growth factor(FGF), and angiopoietin(Ang)] which played a major role in promoting wound angiogenesis in the treatment of DU to provide ideas for further research and new methods for clinical treatment of DU.
Humans ; Medicine, Chinese Traditional ; Ulcer ; Vascular Endothelial Growth Factor A/metabolism* ; Diabetes Complications/drug therapy* ; Wound Healing/physiology* ; Diabetes Mellitus

The present study aimed to investigate the effect of diosgenin on mammalian target of rapamycin(mTOR), fatty acid synthase(FASN), hypoxia inducible factor-1α(HIF-1α), and vascular endothelial growth factor A(VEGFA) expression in liver tissues of rats with non-alcoholic fatty liver disease(NAFLD) and explore the mechanism of diosgenin on lipogenesis and inflammation in NAFLD. Forty male SD rats were divided into a normal group(n=8) fed on the normal diet and an experimental group(n=32) fed on the high-fat diet(HFD) for the induction of the NAFLD model. After modeling, the rats in the experimental group were randomly divided into an HFD group, a low-dose diosgenin group(150 mg·kg~(-1)·d~(-1)), a high-dose diosgenin group(300 mg·kg~(-1)·d~(-1)), and a simvastatin group(4 mg·kg~(-1)·d~(-1)), with eight rats in each group. The drugs were continuously given by gavage for eight weeks. The levels of triglyceride(TG), total cholesterol(TC), low-density lipoprotein cholesterol(LDL-C), alanine transaminase(ALT), and aspartate transaminase(AST) in the serum were detected by the biochemical method. The content of TG and TC in the liver was detected by the enzyme method. Enzyme-linked immunosorbent assay(ELISA) was used to measure interleukin 1β(IL-1β) and tumor necrosis factor α(TNF-α) in the serum. Lipid accumulation in the liver was detected by oil red O staining. Pathological changes of liver tissues were detected by hematoxylin-eosin(HE) staining. The mRNA and protein expression levels of mTOR, FASN, HIF-1α, and VEGFA in the liver of rats were detected by real-time fluorescence-based quantitative polymerase chain reaction(PCR) and Western blot, respectively. Compared with the normal group, the HFD group showed elevated body weight and levels of TG, TC, LDL-C, ALT, AST, IL-1β, and TNF-α(P<0.01), increased lipid accumulation in the liver(P<0.01), obvious liver steatosis, up-regulated mRNA expression levels of mTOR, FASN, HIF-1α, and VEGFA(P<0.01), and increased protein expression levels of p-mTOR, FASN, HIF-1α, and VEGFA(P<0.01). Compared with the HFD group, the groups with drug treatment showed lowered body weight and levels of TG, TC, LDL-C, ALT, AST, IL-1β, and TNF-α(P<0.05, P<0.01), reduced lipid accumulation in the liver(P<0.01), improved liver steatosis, decreased mRNA expression levels of mTOR, FASN, HIF-1α, and VEGFA(P<0.05, P<0.01), and declining protein expression levels of p-mTOR, FASN, HIF-1α, and VEGFA(P<0.01). The therapeutic effect of the high-dose diosgenin group was superior to that of the low-dose diosgenin group and the simvastatin group. Diosgenin may reduce liver lipid synthesis and inflammation and potentiate by down-regulating the mTOR, FASN, HIF-1α, and VEGFA expression, playing an active role in preventing and treating NAFLD.
Rats ; Male ; Animals ; Non-alcoholic Fatty Liver Disease/metabolism* ; Vascular Endothelial Growth Factor A/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Cholesterol, LDL ; Rats, Sprague-Dawley ; Liver ; Inflammation/metabolism* ; Diet, High-Fat/adverse effects* ; TOR Serine-Threonine Kinases/metabolism* ; RNA, Messenger/metabolism* ; Body Weight ; Mammals

Healthy birth and child development are the prerequisite for improving the overall quality of the population. However, premature ovarian failure(POF) threatens the reproductive health of women. The incidence of this disease has been on the rise, and it tends to occur in the young. The causes are complex, involving genetics, autoimmune, infectious and iatrogenic factors, but most of the causes remain unclear. At the moment, hormone replacement therapy and assisted reproductive technology are the main clinical approaches. According to traditional Chinese medicine(TCM), kidney deficiency and blood stasis are one of the major causes of POF, and TCM with the effects of tonifying kidney and activating blood has a definite effect. Through clinical trials, TCM prescriptions for POF have excellent therapeutic effect as a result of multi-target regulation and slight toxicity. In particular, they have no obvious side effects. A large number of studies have shown that the kidney-tonifying and blood-activating TCM can regulate the neuroendocrine function of hypothalamic-pituitary-ovarian axis, improve ovarian hemodynamics and microcirculation, reduce the apoptosis of granulosa cells, alleviate oxidative stress injury, and modulate immunologic balance. The mechanism is that it regulates the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt), vascular endothelial growth factor(VEGF), transforming growth factor(TGF)-β/Smads, nuclear factor E2-related factor 2(Nrf2)/antioxidant response element(ARE), and nuclear factor-kappa B(NF-κB) signaling pathways. This article summarized the pathological mechanisms of tonifying kidney and activating blood TCM in the prevention and treatment of POF and explored the biological basis of its multi-pathway and multi-target characteristics in the treatment of this disease. As a result, this study is expected to serve as a reference for the treatment of POF with the tonifying kidney and activating blood therapy.
Child ; Humans ; Female ; Primary Ovarian Insufficiency/drug therapy* ; Phosphatidylinositol 3-Kinases/metabolism* ; Vascular Endothelial Growth Factor A ; Medicine, Chinese Traditional ; NF-kappa B ; Kidney

The purpose of this study was to explore the effect and mechanism of Xihuang Pills on rats with precancerous lesions of the breast. Of 48 healthy female rats, 8 were randomly selected as blank group, and the other 40 were treated with 7,12-dimethylbenzanthracene(DMBA) combined with estrogen and progestin to establish a model of precancerous lesions of the breast. The successfully modeled rats were randomly divided into a model group, a tamoxifen group(1.8 mg·kg~(-1)·d~(-1)), a Xihuang Pills low-dose group(0.3 g·kg~(-1)·d~(-1)), a medium-dose group(0.6 g·kg~(-1)·d~(-1)) and a high-dose group(1.2 g·kg~(-1)·d~(-1)). After 30 days of admi-nistration, the histopathological changes of viscera and breast were observed by haematoxylin and eosin(HE) staining, and the visceral index was calculated. Enzyme linked immunosorbent assay(ELISA) was used to detect the contents of estradiol(E_2) and progesterone(P) in serum. The protein expressions of vascular endothelial growth factor(VEGF) and fibroblast growth factor 2(FGF2) were detected by immunohistochemistry. The protein expressions of VEGF, vascular endothelial growth factor receptor 2(VEGFR2), phosphorylated-vascular endothelial growth factor receptor 2(p-VEGFR2), B-cell lymphoma-2(Bcl-2), and Bcl-2 associated X protein(Bax) were detected by Western blot and the mRNA expressions of VEGF, FGF2, CXC-chemokine receptor 4(CXCR4), cysteine aspartic acid-specific protease(caspase-3), and stromal cell-derived factor 1(SDF-1) were detected by real-time polymerase chain reaction(RT-PCR). HE staining revealed that the model group had some liver and kidney damages and severe hyperplastic mammary tissue, while the Xihuang Pills high-dose group had mild hyperplasia. Compared with the model group, the Xihuang Pills groups had lo-wer ovarian coefficient(P<0.05 or P<0.01) and Xihuang Pills high-dose group had lower uterine coefficient(P<0.01). ELISA results showed that compared with the model group, expressions of E_2 and P in Xihuang Pills high-dose group were significantly decreased(P<0.05 or P<0.01). Immunohistochemistry, Western blot and RT-PCR indicated that compared with the conditions in the model group, the protein and mRNA expressions of VEGF and FGF2 in the Xihuang Pills groups were down-regulated(P<0.05 or P<0.01), and the protein expression of Bcl-2 was lowered(P<0.01); there was a decrease in the protein expressions of VEGFR2 and p-VEGFR2(P<0.01), a down-regulation in the mRNA expressions of CXCR4 and SDF-1(P<0.01), while an increase in the mRNA expression of caspase-3(P<0.01) in both Xihuang Pills medium-dose and high-dose groups; the protein expression of Bax in Xihuang Pills high-dose group was increased(P<0.01). The above results indicated that Xihuang Pills can effectively intervene in precance-rous lesions of the breast, and the mechanism may be related to the regulation of E_2 and P secretion as well as the inhibition of angiogenesis and chemokine receptor expression, thus controlling the occurrence of precancerous lesions of the breast in rats.
Rats ; Female ; Animals ; Rats, Sprague-Dawley ; bcl-2-Associated X Protein ; Vascular Endothelial Growth Factor A/metabolism* ; Caspase 3 ; Vascular Endothelial Growth Factor Receptor-2 ; Fibroblast Growth Factor 2 ; Proto-Oncogene Proteins c-bcl-2 ; 9,10-Dimethyl-1,2-benzanthracene/toxicity* ; Precancerous Conditions ; Hyperplasia ; Receptors, Chemokine ; RNA, Messenger

Objective To investigate the ameliorative effect of salidroside on diabetes retinopathy (DR) rats and its mechanism. Methods Male SD rats were randomly divided into blank group, model group, low-dose and high-dose salidroside treatment groups. Except for the blank group, other groups were modeled by intraperitoneal injection of streptozotocin. After successful modeling, treatment groups were injected intraperitoneally with [50 mg/(kg.d)] and [100 mg/(kg.d)] salidroside respectively, for 4 weeks; the blank group and model group were injected with corresponding doses of saline. ELISA was used to measure the expression levels of antioxidant-related enzyme activity and inflammatory factors in blood glucose and serum of rats in each group. Retinal tissue lesions were detected by HE staining, and the expression of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule 1 (ICAM-1) in retinal tissues were detected by immunohistochemical staining. Western blot analysis was used to detect the expression of phosphatidylinositol 3 kinase (PI3K) , nuclear factor κB p65 (NF-κB p65), phosphorylated p38 MAPK (p-p38 MAPK), and phosphorylated protein kinase B (p-AKT) proteins. Results Compared with model group, salidroside could significantly reduce blood glucose level and increase body mass in DR rats. The serum levels of superoxide dismutase (SOD) and catalase (CAT) were significantly increased, while the levels of malondialdehyde (MDA), tumor necrosis factor α (TNF-α), interleukin 6 (IL-6) and IL-1β were reduced. The protein expression of VEGF, ICAM-1, NF-κB p65 and p-p38 MAPK was significantly decreased, while the protein expression of PI3K and p-AKT was increased. Conclusion Salidroside can reduce DR in rats by inhibiting oxidative stress and immune inflammatory response, which may be related to the reduction of abnormal expression of VEGF and ICAM-1 and the activation of PI3K/AKT signaling pathway.
Animals ; Male ; Rats ; Blood Glucose ; Diabetes Mellitus ; Inflammation/metabolism* ; Intercellular Adhesion Molecule-1/metabolism* ; NF-kappa B/metabolism* ; Oxidative Stress ; Phosphatidylinositol 3-Kinases/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Rats, Sprague-Dawley ; Retinal Diseases ; Tumor Necrosis Factor-alpha/metabolism* ; Vascular Endothelial Growth Factor A/metabolism*

OBJECTIVE: To investigate the anti-angiogenic activity of Kunxian Capsule (KX) extract and explore the underlying molecular mechanism using zebrafish. METHODS: The KX extract was prepared with 5.0 g in 100 mL of 40% methanol followed by ultrasonication and freeze drying. Freeze dried KX extract of 10.00 mg was used as test stock solution. Triptolide and icariin, the key bioactive compounds of KX were analyzed using ultra-high performance liquid chromatography. The transgenic zebrafish Tg(flk1:GFP) embryos were dechorionated at 20-h post fertilization (hpf) and treated with PTK 787, and 3.5, 7, 14 and 21 µg/mL of KX extract, respectively. After 24-h post exposure (hpe), mortality and malformation (%), intersegmental vessels (ISV) formation, and mRNA expression level of angiogenic pathway genes including phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), extracellular signal-regulated kinases (ERKs), mitogen-activated protein kinase (MAPK), vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF-2) were determined. Further, the embryos at 72 hpf were treated with KX extract to observe the development of sub-intestinal vein (SIV) after 24 hpe. RESULTS: The chromatographic analysis of test stock solution of KX extract showed that triptolide and icariin was found as 0.089 mg/g and 48.74 mg/g, respectively, which met the requirements of the national drug standards. In zebrafish larvae experiment, KX extract significantly inhibited the ISV (P<0.01) and SIV formation (P<0.05). Besides, the mRNA expression analysis showed that KX extract could significantly suppress the expressions of PI3K and AKT, thereby inhibiting the mRNA levels of ERKs and MAPK. Moreover, the downstream signaling cascade affected the expression of VEGF and its receptors (VEGFR and VEGFR-2). FGF-2, a strong angiogenic factor, was also down-regulated by KX treatment in zebrafish larvae. CONCLUSION KX extract exhibited anti-angiogenic effects in zebrafish embryos by regulating PI3K/AKT-MAPK-VEGF pathway and showed promising potential for RA treatment.
Animals ; Fibroblast Growth Factor 2 ; Human Umbilical Vein Endothelial Cells ; Mitogen-Activated Protein Kinases/metabolism* ; Phosphatidylinositol 3-Kinase ; Phosphatidylinositol 3-Kinases/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Vascular Endothelial Growth Factor A/metabolism* ; Zebrafish

Mesenchymal stem cell (MSC)-derived exosomes (Exos) were reported to a prospective candidate in accelerating diabetic wound healing due to their pro-angiogenic effect. MSCs pretreated with chemistry or biology factors were reported to advance the biological activities of MSC-derived exosomes. Hence, this study was designed to explore whether exosomes derived from human umbilical cord MSCs (hucMSCs) preconditioned with Nocardia rubra cell wall skeleton (Nr-CWS) exhibited superior proangiogenic effect on diabetic wound repair and its underlying molecular mechanisms. The results showed that Nr-CWS-Exos facilitated the proliferation, migration and tube formation of endothelial cells in vitro. In vivo, Nr-CWS-Exos exerted great effect on advancing wound healing by facilitating the angiogenesis of wound tissues compared with Exos. Furthermore, the expression of circIARS1 increased after HUVECs were treated with Nr-CWS-Exos. CircIARS1 promoted the pro-angiogenic effects of Nr-CWS-Exos on endothelial cellsvia the miR-4782-5p/VEGFA axis. Taken together, those data reveal that exosomes derived from Nr-CWS-pretreated MSCs might serve as an underlying strategy for diabetic wound treatment through advancing the biological function of endothelial cells via the circIARS1/miR-4782-5p/VEGFA axis.
Humans ; Endothelial Cells/metabolism* ; Exosomes/metabolism* ; Cell Wall Skeleton/metabolism* ; Neovascularization, Physiologic ; Wound Healing/physiology* ; MicroRNAs/metabolism* ; Diabetes Mellitus ; Vascular Endothelial Growth Factor A/metabolism*