Protective effect of alizarin on vascular endothelial dysfunction via inhibiting the type 2 diabetes-induced synthesis of THBS1 and activating the AMPK signaling pathway.

BACKGROUND: In this study, we investigated the protective effects of alizarin (AZ) on endothelial dysfunction (ED). AZ has inhibition of the type 2 diabetes mellitus (T2DM)-induced synthesis of thrombospondin 1 (THBS1). Adenosine 5'-monophosphate- activated protein kinase (AMPK), particularly AMPKα2 isoform, plays a critical role in maintaining cardiac homeostasis. PURPOSE: The aim of this study was to investigate the ameliorative effect of AZ on vascular injury caused by T2DM and to reveal the potential mechanism of AZ in high glucose (HG)-stimulated human umbilical vein endothelial cells (HUVECs) and diabetic model rats. STUDY DESIGN: HUVECs, rats and AMPK-/- transgenic mice were used to investigate the mitigating effects of AZ on vascular endothelial dysfunction caused by T2DM and its in vitro and in vivo molecular mechanisms. METHODS: In type 2 diabetes mellitus rats and HUVECs, the inhibitory effect of alizarin on THBS1 synthesis was verified by immunohistochemistry (IHC), immunofluorescence (IF) and Western blot (WB) so that increase endothelial nitric oxide synthase (eNOS) content in vitro and in vivo. In addition, we verified protein interactions with immunoprecipitation (IP). To probe the mechanism, we also performed AMPKα2 transfection. AMPK's pivotal role in AZ-mediated prevention against T2DM-induced vascular endothelial dysfunction was tested using AMPKα2-/- mice. RESULTS: We first demonstrated that THBS1 and AMPK are targets of AZ. In T2DM, THBS1 was robustly induced by high glucose and inhibited by AZ. Furthermore, AZ activates the AMPK signaling pathway, and recoupled eNOS in stressed endothelial cells which plays a protective role in vascular endothelial dysfunction. CONCLUSIONS: The main finding of this study is that AZ can play a role in different pathways of vascular injury due to T2DM. Mechanistically, alizarin inhibits the increase in THBS1 protein synthesis after high glucose induction and activates AMPKα2, which increases NO release from eNOS, which is essential in the prevention of vascular endothelial dysfunction caused by T2DM.

Protective effect of vitamin B6 against doxorubicin-induced cardiotoxicity by modulating NHE1 expression.

Doxorubicin (DOX) has been used to treat various types of cancer, but its application is limited due to its heart toxicity as well as other drawbacks. Chronic inhibition of Na+ /H+ exchanger (NHE1) reduces heart failure and reduces the production of reactive oxygen species (ROS); vitamin B6 (VitB6 ) has been demonstrated to have a crucial role in antioxidant mechanism. So, this study was designed to explore the effect of VitB6 supplement on the DOX-induced cardiotoxicity and to imply whether NHE1 is involved. Ultrasonic cardiogram analysis revealed that VitB6 supplement could alleviate DOX-induced cardiotoxicity; hematoxylin and eosin (HE) and Masson's staining further confirmed this effect. Furthermore, VitB6 supplement exhibited significant antioxidative stress and antiapoptosis effect, which was evidenced by decreased serum malondialdehyde (MDA) content and increased serum superoxide dismutase (SOD) content, and decreased Bcl-2-associated X protein/B-cell lymphoma-2 ratio, respectively. Collectively, VitB6 supplement may exert antioxidative and antiapoptosis effects to improve cardiac function by decreasing NHE1 expression and improve DOX-induced cardiotoxicity.

Diagnostic ultrasound-mediated microbubble cavitation dose-dependently improves diabetic cardiomyopathy through angiogenesis.

Ultrasound-mediated microbubble cavitation (UMMC) induces therapeutic angiogenesis to treat ischemic diseases. This study aimed to investigate whether diagnostic UMMC alleviates diabetic cardiomyopathy (DCM) and, if so, through which mechanisms. DCM model was established by injecting streptozocin into rats to induce hyperglycemia, followed by a high-fat diet. The combined therapy of cation microbubble with low-intensity diagnostic ultrasound (frequency = 4 MHz), with a pulse frequency of 20 Hz and pulse length (PL) of 8, 18, 26, or 36 cycles, was given to rats twice a week for 8 consecutive weeks. Diagnostic UMMC therapy with PL at 8, 18, and 26 cycles, but not 36 cycles, dramatically prevented myocardial fibrosis, improved heart functions, and increased angiogenesis, accompanied by increased levels of PI3K, Akt, and eNOS proteins in the DCM model of rats. In cultured endothelial cells, low-intensity UMMC treatment (PL = 3 cycles, sound pressure level = 50%, mechanical index = 0.82) increased cell viability and activated PI3K-Akt-eNOS signaling. The combination of diagnostic ultrasound with microbubble destruction dose-dependently promoted angiogenesis, thus improving heart function through PI3K-Akt-eNOS signaling in diabetes. Accordingly, diagnostic UMMC therapy should be considered to protect the heart in patients with diabetes.

Nitrosative stress induced by homocysteine thiolactone drives vascular cognitive impairments via GTP cyclohydrolase 1 S-nitrosylation in vivo.

BACKGROUND: s: Hyperhomocysteinemia (HHcy) is one of risk factors for vascular cognitive impairment (VCI). GTP cyclohydrolase 1 (GCH1) deficiency is critical to oxidative stress in vascular dysfunction. The aim of this study was designed to examine whether HHcy induces VCI through GCH1 S-nitrosylation, a redox-related post-translational modification of cysteine. METHODS: The VCI model was induced by feeding mice homocysteine thiolactone (HTL) for 16 consecutive weeks. The cognitive functions were evaluated by step-down avoidance test, passive avoidance step-through task test, and Morris water maze (MWM) test. Protein S-nitrosylation was assayed using a biotin-switch method. RESULTS: In cell-free system, nitric oxide (NO) donor induced GCH1 protein S-nitrosylation and decreased GCH1 activity. In endothelial cells, HTL increased GCH1 S-nitrosylation, reduced tetrahydrobiopterin, and induced oxidative stress, which were attenuated by N-acetyl-cysteine, L-N6-1-Iminoethyl-lysine, mutant of GCH1 cysteine 141 to alanine (MT-GCH1) or gene deletion of inducible NO synthase (iNOS). Further, HTL incubation or iNOS overexpression promoted endothelial cellular senescence, but abolished by exogenous expression of MT-GCH1 or pharmacological approaches including N-acetyl-cysteine, L-sepiapterin, and tempol. In wildtype mice, long-term administration of HTL induced GCH1 S-nitrosylation and vascular stiffness, decreased cerebral blood flow, and damaged the cognitive functions. However, these abnormalities induced by HTL administration were rescued by enforced expression of MT-GCH1 or gene knockout of iNOS. In human subjects, GCH1 S-nitrosylation was increased and cognitive functions were impaired in patients with HHcy. CONCLUSION: The iNOS-mediated nitrosative stress induced by HTL drives GCH1 S-nitrosylation to induce cerebral vascular stiffness and cognitive impairments.

Tert-Butylhydroquinone alleviates insulin resistance and liver steatosis in diabetes.

OBJECTIVES: This work aimed to determine tert-Butylhydroquinone (TBHQ)'s effects on insulin resistance (IR) and liver steatosis in diabetic animals and to explore the underpinning mechanisms. MATERIALS AND METHODS: Male ApoE-/-mice underwent streptozocin (STZ) administration while receiving a sucrose/fat-rich diet for type 2 diabetes mellitus (T2DM) establishment. This was followed by a 6-week TBHQ administration. Body weight, fasting (FBG) and postprandial (PBG) blood glucose amounts, and insulin concentrations were measured, and the oral glucose tolerance test (OGTT) was carried out. Hematoxylin and eosin (H and E) staining and immunoblot were carried out for assessing histology and protein amounts in the liver tissue samples. In addition, cultured HepG2 cells were administered HClO and insulin for IR induction, and immunoblot was carried out for protein evaluation. Finally, the cells were stained with the Hoechst dye for apoptosis evaluation. RESULTS: The model animals showed T2DM signs, and TBHQ decreased FBG, ameliorated glucose tolerance and reduced liver steatosis in these animals. In addition, TBHQ markedly upregulated AMPKα2, GLUT4 and GSK3 ß, as well as phosphorylated PI3K and AKT in the liver of mice with T2DM. In agreement, TBHQ decreased HClO-and insulin-related IR in cells and suppressed apoptosis through AMPKα2/PI3K/AKT signaling. CONCLUSIONS: TBHQ alleviates IR and liver steatosis in a mouse model of T2DM likely through AMPKα2/PI3K/AKT signaling.

Dok3 is involved in cisplatin-induced acute kidney injury via regulation of inflammation and apoptosis.

Cisplatin-induced acute kidney injury (AKI) is associated with high morbidity and mortality worldwide, but the underlying mechanisms are not fully understood. Downstream-of-kinase 3 (Dok3), a member of the Dok family of adaptor proteins plays a critical role in inflammatory response and immune regulation; however, the role of Dok3 in cisplatin-induced AKI remains unclear. This study explored the effect and potential molecular mechanisms of Dok3 in cisplatin-induced AKI using Dok3 knockout (Dok3-/-) and control mice (129S) with or without administration of a single intraperitoneal injection of cisplatin. Apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, lactate dehydrogenase (LDH) release, and Hoechst staining. Inflammatory factors were measured using ELISA kits. Protein and gene expression levels were measured by western blot analysis and real-time PCR, respectively. The results showed that Dok3 was expressed in renal tubular epithelial cells. Dok3 expression was decreased in kidneys of mice treated with cisplatin and cisplatin-treated HK2 cells. Dok3-/- mice showed lower creatinine levels and NGAL expression, and increased survival rates compared to 129S mice. Cisplatin-induced production of TNF-α and IL-6, and renal tubular cell apoptosis was attenuated in Dok3-/- mice. In vitro experiments demonstrated that HK2 cells overexpressing Dok3 exhibited exacerbated cisplatin-induced apoptosis and production of TNF-α and IL-6. These findings demonstrate that Dok3 regulates cisplatin-induced AKI by regulating apoptosis and inflammation.

The antihypertension effect of hydrogen sulfide (H2S) is induced by activating VEGFR2 signaling pathway.

AIMS: Previous studies demonstrated that H2S has an antihypertension effect on hypertension, but the mechanism involved is unclear until now. The aim of the study is to elucidate the effect of H2S on PH and the mechanism involved. MAIN METHODS: In this study, GYY4137 (a H2S donor) were administered to spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) by intraperitoneally injection daily for consecutive 14 days. Systolic blood pressure (SBP), endothelial-dependent relaxation (EDR), plasma malondialdehyde (MDA), superoxide dismutase (SOD), and H2S levels were measured. Human umbilical vein endothelial cells (HUVECs) were also used to elucidate the mechanism involved in the protect effect of H2S on the injured vessels. KEY FINDINGS: Our results showed that GYY4137 normalized the SBP (P < 0.0001), increased EDR (P < 0.01), reduced oxidative stress (increased the content of SOD and reduced the content of MDA) of SHR. Meanwhile, GYY4137 could promote the proliferation (P < 0.01) and migration (P < 0.01) of HUVECs, increase the expression of endothelial NO synthase (eNOS) and Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) both in SHR and HUVECs treated with GYY4137. In addition to the above results, the PIP3/Akt signaling pathway was activated and the expression of caspase 3 was increased by GYY4137. However, all the above effects of GYY4137 were blocked by ZD6474 (a VEGFR2 inhibitor). SIGNIFICANCE: GYY4137 had a hypotensive and vascular protect effect on PH. This effect might be mediated through upregulating the expression of VEGFR2, which subsequently alleviating oxidant-provoked vascular endothelial dysfunction, and promoting the proliferation and migration of endothelial cells in SHR.

MicroRNA-140-5p targeting tumor necrosis factor-α prevents pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is characterized by the apoptosis resistance and hyperproliferation of pulmonary artery smooth muscle cells (PASMCs). Its pathogenesis has not been revealed. Here, we carried out experiments to investigate the functions of miR-140-5p and tumor necrosis factor-α (TNF-α). METHODS: We selected GSE703 from Gene Expression Omnibus (GEO) Database to conduct microarray analysis using R software and Gene Set Enrichment Analysis (GSEA). Combing bioinformatics results, the upregulation of miR-140-5p inhibited PAH progression through targeting TNF-α. RNA expression was measured by quantitative real-time polymerase chain reaction (RT-qPCR) and protein level was measured by western blot analysis and enzyme-linked immunosorbent assays (ELISA). We conducted monocrotaline (MCT) injection to rats to form PAH animal models. The lung tissues were observed by hematoxylin-eosin (HE) staining and Sirius red-picric acid staining. Right ventricular systolic pressure (RVSP) and the ratio of right ventricle (RV)-to-left ventricle (LV) plus septum (S) weight (RV/[LV + S]) were measured in MCT-induced animal models. Overexpression of miR-140-5p and TNF-α were utilized to research the proliferation, migration, and phenotypic variation of hypoxia-mediated PASMCs. The binding between miR-140-5p and TNF-α 3'-untranslated region (3'-UTR) was confirmed via luciferase reporter assay. RESULTS: Downregulation of miR-140-5p and upregulation of TNF-α were observed in PAH rat model and hypoxia-mediated PASMCs. And we proved that overexpression of miR-140-5p could suppress the proliferation, migration, and phenotypic variation of PASMCs, therefore inhibiting PAH pathogenesis. Luciferase assay verified that miR-140-5p targeted TNF-α directly. A converse correlation was also shown between miR-140-5p and TNF-α in PASMCs. CONCLUSIONS: miR-140-5p and TNF-α are important regulators in PAH pathology and may serve as a therapeutic target for PAH.

Berberine promotes ischemia-induced angiogenesis in mice heart via upregulation of microRNA-29b.

BACKGROUND: Berberine has several preventive effects on cardiovascular diseases. Increased expression of miR-29b has been reported to attenuate cardiac remodeling after myocardial infarction (MI). We hypothesized that berberine via an miR-29b-dependent mechanism promotes angiogenesis and improves heart functions in mice after MI. METHODS: The MI model was established in mice by ligation of left anterior descending coronary artery. The expression of miR-29b was examined by RT-qPCR. Angiogenesis was assessed by immunohistochemistry. RESULTS: Berberine increased miR-29b expression and promoted cell proliferations and migrations in cultured endothelial cells, which were abolished by miR-29b antagomir or AMP-activated protein kinase inhibitor compound C. In mice following MI, administration of berberine significantly increased miR-29b expressional level, promoted angiogenesis, reduced infarct size, and improved heart functions after 14 postoperative days. Importantly, these in vivo effects of berberine were ablated by antagonism of miR-29b. CONCLUSION: Berberine via upregulation of miR-29b promotes ischemia-induced angiogenesis and improves heart functions.

Inhibition of NA(+)/H(+) Exchanger 1 Attenuates Renal Dysfunction Induced by Advanced Glycation End Products in Rats.

It has been recognized that sodium hydrogen exchanger 1 (NHE1) is involved in the development of diabetic nephropathy. The role of NHE1 in kidney dysfunction induced by advanced glycation end products (AGEs) remains unknown. Renal damage was induced by AGEs via tail vein injections in rats. Function and morphology of kidney were determined. Compared to vehicle- or BSA-treated rats, AGEs caused abnormalities of kidney structures and functions in rats, accompanied with higher MDA level and lower GSH content. Gene expressions of NHE1 gene and TGF-ß1 in the renal cortex and urine were also increased in AGEs-injected rats. Importantly, all these detrimental effects induced by AGEs were reversed by inhibition of NHE1 or suppression of oxidative stress. These pieces of data demonstrated that AGEs may activate NHE1 to induce renal damage, which is related to TGF-ß1.

NK3 and NK4 of HGF enhance filamin production via STAT pathway, but not NK1 and NK2 in human breast cancer cells.

AIM: The purpose of this study was to reveal the effects of hepatocyte growth factor (HGF) variants on human breast cancer cells and the differential signaling pathways of the variants in controlling cell proliferation and invasion. METHODS: Four HGF variants (NK1, NK2, NK3, and NK4) were created by gene engineering, and the variant DNA fragments were cloned into pGEM-T for DNA sequencing and then transferred to a pTrcHis-A plasmid for expression. Recombinant proteins were purified from Escherichia coli, and a series of assays, including cell proliferation and invasion were carried out. Phosphorylated components in the HGF-c-Met and STAT (signal transducers and activators of transcription) pathways were detected by immunoprecipitation-Western blots. RESULTS: All the HGF variants inhibited the vigorous growth of the cancer cells differently and dose-dependently, but the effect of NK3 or NK4 was 7.5-fold higher than NK1 or NK2. In addition, the assays for the phosphorylation of the components in the HGF-c-Met pathway showed that NK3 and NK4 inhibited invasion via the STAT pathway, whereas NK1 and NK2 were via the HGF-c-Met pathway. CONCLUSION: The engineered HGF variants inhibited the proliferation of human breast cancer cells via different signaling pathways, NK1 and NK2 via the HGF-c-Met pathways, and NK3 and NK4 via the STAT pathway, the latter being a possible key route for the inhibition of cell invasion. All of the HGF variants have the potential to become pharmaceutical drugs in the treatment of human cancer.

[Proliferation and differentiation-inducing effects of aminosteroid H42648 on K562 leukemia cells].

OBJECTIVE: To explore the proliferation and differentiation-inducing effects of aminosteroid H42648 on K562 leukemia cells. METHODS: The effects of H42648 on K562 cell proliferation were determined by cell count, colony count and MTT assay, and the differentiation by morphology, benzidine stain and flow cytometry. RESULTS: The growth of K562 cells was inhibited after the treatment with H42648 for 5 days. The inhibition rates for K562 cells in MTT assay were (26.90+/-3.18)% and (43.26+/-2.54)% after the treatment by 10(-8)mol/L and 10(-6)mol/L H42648. The inhibitions for K562 cells in colony culture assay were (22.21+/-9.20)% and (48.71+/-8.24)% at 10(-8)mol/L and 10(-6)mol/L H42648, respectively. H42648 also induced K562 cells toward erythroid differentiation. It was verified by benzidine stain that the OD value at 10(-8)mol/L H42648 was increased by (153+/-17.65)% compared with the control, and at 10(-6)mol/L H42648 the OD value was increased by (250+/-4.60)% compared with the control. The morphology showed the differentiation tendency of K562 cells after the treatment. Data from flow cytometry showed that after the treatment with 10(-6)mol/L H42648 for 4 days, positive CD71 expression in differentiated K562 cells was 89.91%. CONCLUSION: H42648 can inhibit the proliferation of K562 cell lines and induce toward erythroid differentiation. H42648 may become a new lead for the differentiation-inducing therapy for leukemia.